POST-HARVEST TRANSMISSION OF Salmonella enterica TO THE ROOTS AND

LEAVES OF INTACT PACKAGED BUTTERHEAD LETTUCE

By

Jessie A. Waitt

Thesis submitted to the faculty of the Virginia Polytechnic Institute and State University in

partial fulfillment of the requirements for the degree of

Master of Science in Life Science

in

Food Science and Technology

Monica A. Ponder, Chair

Gregory E. Welbaum

David D. Kuhn

April 22, 2013

Blacksburg, Virginia

Keywords: Living lettuce, Salmonella enterica, hydroponic production

POST-HARVEST TRANSMISSION OF Salmonella enterica TO THE ROOTS AND

LEAVES OF INTACT PACKAGED BUTTERHEAD LETTUCE

Jessie A. Waitt

ABSTRACT

In the United States, illnesses associated with fresh produce are increasing in frequency. While

contamination risks are present at every aspect of the farm to fork continuum, post-harvest

practices holds the potential for cross-contamination of large amounts of product. Post-harvest

contamination risks for hydroponically grown lettuce packaged with intact roots and sold as

‘living lettuce’ are poorly understood. In this study, transmission of Salmonella enterica

serotype Enteritidis to the roots and leaves of butterhead lettuce was studied when contamination

was introduced during typical handling practices. The effectiveness of random sampling

strategies for selection of Salmonella contaminated leaves was assessed by co-inoculating the

Salmonella solution with Glo Germ™ and comparing recovery from blacklight selected leaves.

The recovery of Salmonella was improved by only 0.5 log CFU/g when blacklight was used to

select Glo Germ™ contaminated leaves (P=0.05). This suggests random leaf selection as

described by current FDA protocols is adequate. In addition, this study showed rapid transfer of

Salmonella from liquid to the roots and sub-sequentially to the leaves of living lettuce.

Salmonella persisted but did not grow on leaves when stored at 4˚C for 18-days. Storage at 12˚C

was associated with 2 log CFU/g increases in Salmonella on roots after 18-days storage

(P=0.0002), while 4˚C storage was associated with a decrease of 0.4 log CFU/g Salmonella on

roots (P=0.0001). Growth occurred only under temperature abuse conditions. This reinforces the

need for maintaining temperature control and highlights the importance of identifying risks

associated with post-harvest handling during hydroponic production and distribution.

iii

ACKNOWLEDGMENTS

First, I would like to recognize my advisor, Dr. Monica Ponder, for her endless patience and

efforts in helping me complete my thesis. It would have not been possible without her creative

teaching styles and constant encouragement to keep me aligned. It has been a pleasure working

with you Dr. Ponder.

I would also like to extend my appreciation to the other committee members, Dr. David Kuhn

and Dr. Gregory Welbaum for their expertise and providing positive feedback towards the

completion of my thesis work.

To my lab mate, Courtney Klotz, thank you for making my lab projects more enjoyable. I will

always be more than happy to “hold your needle” in whatever situation life may bring you.

To the Food Science and Technology staff and faculty members, thank you for providing

accommodations to ensure that I will have the best learning experience. Thank you for giving me

the privilege to continue my strong desires of learning in the completion of my thesis and

coursework.

To my family in England and New Zealand, you are never too far from home but forever close in

my heart. Thank you for the constant cheering even when the finish line seemed so far away.

To my horse, Miss Tarzana, thank you for being my best friend. Your devotion and sweet love

has made every ride therapeutic. Thank you for the valuable lessons that my grandparents or

instructors could not teach me. Thank you for carrying all of my burdens and somehow always

find a way to put a smile on my face even on my weakest days. It has been an incredible journey

and I look forward to many more rides with you.

To my sweetheart, Michael Miller, there are no words to describe the appreciation and sacrifices

you have done to keep our relationship strong. I am truly blessed to have you in my life and to

stand beside me in whatever life brings us. Thanks for always loving me even at my worse and I

look forward spending the rest of my life with you.

Last but not least, to my grandparents, thank you for being the best parents even when you did

not have to be. Life has not always been kind and thank you for restoring faith that there is

goodness in life. Most of all, I hope I have made you proud every step of the way.

iv

DEDICATION

I dedicate this work to my grandparents, James F. and Suzanne B. Fiscus, for teaching me that I

can do anything except hear, to never let go of your dreams, and that hard work truly does have

its rewards.

v

ATTRIBUTION

Several people contributed to the completion of the research described in this thesis,

and a description of their contributions are included below.

Monica A. Ponder- Ph.D. (Department of Food Science and Technology, Virginia Tech) is the

primary advisor and head of the committee chair. Dr. Ponder provided positive guidance in the

development of this research project, and resources to fund this study. Her background

knowledge in food safety and food microbiology significantly contributed to this project.

David D. Kuhn- Ph.D. (Department of Food Science and Technology, Virginia Tech) was an

active member of the committee and provided expertise and support in hydroponic system set-up

at the Southwest Virginia Aquaculture Research Center , Virginia. His mentorship and

knowledge greatly contributed to this research project.

Gregory E. Welbaum-Ph.D. (Department of Horticulture, Virginia Tech) was an active member

of the committee and provided expertise in horticultural crops and assistance in the living lettuce

production. His mentorship and knowledge greatly contributed to this research work.

Daniel Taylor- (Department of Food Science and Technology, Virginia Tech) provided

background knowledge in designing hydroponic system and was responsible for growing the

butterhead lettuce used in this project at the Southwest Virginia Aquaculture Research Center in

Saltville, Virginia.

vi

TABLE OF CONTEXTS Page

Abstract ........................................................................................................................................... ii

Acknowledgements ........................................................................................................................ iii

Dedication ...................................................................................................................................... iv

Attribution ....................................................................................................................................... v

Table of Contents ........................................................................................................................... vi

List of Tables……………………………………………………………………………..……..viii

List of Figures ................................................................................................................................ ix

Chapter 1: Introduction and Justification ...................................................................................... 1

References………………………………………………………………………………...4

Chapter 2: Literature Review ........................................................................................................ 6

Introduction ......................................................................................................................... 6

Production Value, Per Capita Consumption, and Exported and Importation of Fresh

Lettuce................................................................................................................................. 7

Statistical Analysis of Foodborne Illnesses Outbreaks Burden in the United States .......... 8

Notable Outbreaks Associated with Leafy Greens ............................................................. 9

Characteristics and Morphology of Salmonella enterica .................................................. 12

Pre-Harvest Handling and Potential Reservoirs of Contamination .................................. 14

Post-Harvest Packaging and Handling and Potential Reservoirs of Contamination ........ 16

Molecular Mechanisms of Attachment of Salmonella species to Plants .......................... 18

Food Safety and Prevention Strategies ............................................................................. 20

Methods of Leafy Greens Sanitation ................................................................................ 22

Hydroponics ...................................................................................................................... 23

Hydroponic production of Butterhead Lettuce ................................................................. 25

Post-Harvest Handling of Butterhead Lettuce in a Hydroponic Production..................... 27

Typical Production Practices in Small Hydroponic Greenhouses in Virginia .................. 31

Potential Routes of Transfer of Salmonella species in a Hydroponic Production ............ 34

Glo Germ™ as a Surrogate for Identification of Post-Harvest Cross-Contamination ..... 35

Conclusions ....................................................................................................................... 37

References ......................................................................................................................... 39

Chapter 3: Glo Germ™ as a Surrogate for Identification of Post-Harvest Cross-Contamination

on Living Lettuce .......................................................................................................................... 48

Abstract…………………………………………………………………………………..48

Introduction ....................................................................................................................... 50

vii

Materials and methods ...................................................................................................... 51

Results ............................................................................................................................... 55

Discussion ......................................................................................................................... 56

Conclusion ........................................................................................................................ 61

References ......................................................................................................................... 63

Figures……………………………………………………………………………………66

Chapter 4: Post-harvest Transfer and Survival of Salmonella enterica serotype Enteritidis on

Living Lettuce ............................................................................................................................... 70

Abstract…………………………………………………………………………………..70

Introduction ....................................................................................................................... 71

Materials and Methods ...................................................................................................... 73

Results ............................................................................................................................... 76

Discussion ......................................................................................................................... 76

Conclusion ........................................................................................................................ 81

References ......................................................................................................................... 82

Figures……………………………………………………………………………………86

Chapter 5: Conclusion and Future Research Direction ............................................................... 89

viii

LIST OF TABLES

Page

CHAPTER 2: LITERATURE REVIEW

Table 2.1: The number of people affected by food-borne illnesses, hospitalizations, and

deaths linked to 31 known pathogens in 2000-2008………..……………………………..8

Table 2.2: The number of food-borne outbreaks, illnesses, and deaths cases associated

with leafy greens reported from 1973-2006……………………………………………….9

Table 2.3: Recalls reported from 2004-2012 associated Salmonella with

various salad blends from field production………………………………………………11

Table 2.4: Potential routes of transfer of Salmonella enterica in a hydroponic

production………………………………………………………………………………..34

ix

LIST OF FIGURES Page

CHAPTER 3: Glo Germ™ as a Surrogate for Identification of Post-Harvest

Cross-Contamination on Living Lettuce

Figure 3.1: Red food coloring was used as an indicator to visualize where

the gloves came into contact with the different parts of lettuce plants

during routine packaging of living lettuce.......................................................................66

Figure 3.2: Droplets of Glo Germ™ solution on a lettuce head visualized

using blacklight. A) Living lettuce viewed on its side, showing the distribution

from bottom to top of the leaf. B) View of top of the head of living lettuce,

showing the wide dispersion throughout the whole surface area and inner

leaves…………………………………………………………………………………….66

Figure 3.3: Images of older and outer lettuce leaves used to calculate the average

intensity (Average Quantity) within a 30x30 mm area. The average quantity value of the

leaf without (background) Glo Germ™ was subtracted from the average intensity value

of the leaf with Glo Germ™ to determine the fluorescence only by Glo Germ™………67

Figure 3.4: Enumeration of Salmonella Enteritidis ptvs 177 recovered from

living lettuce leaves chosen using two different selection methods: random

selection and blacklight to visualize Glo-Germ co-inoculated with the Salmonella.

Data shows the average log CFU/g of Salmonella and error bars from 3 replicates

(n=9) and cells were recovered by plating on XLT4-Rif and incubated at

37˚C for 48 h. Each error bar is constructed using 1 standard deviation from the mean.

Samples not connected by same letter are significantly different (P<0.05)...…………...68

Figure 3.5: The recoverability of Salmonella Enteritidis ptvs 177 following a

single contamination event from worker gloves followed by sequential post-harvest

packing of living lettuce using two different selection methods. Data are the

average log CFU/g of Salmonella and error bars from 3 replicates (n=9) and

cells were recovered by plating on XLT4-Rif and incubated at 37˚C for 48 h.

Each error bar is constructed using 1 standard deviation from the mean.

Samples not connected by same letter are significantly different (P<0.05)………..……69

Chapter 4: Post-harvest Transfer and Survival of Salmonella enterica

Serotype Enteritidis on Living Lettuce

Figure 4.1: A series of pictures demonstrating post-harvest handling and

inoculation of the heads. A) Lettuce heads and intact root systems were

harvested at Virginia Tech Aquaculture Research Center Saltville, VA.

B) Post-harvest Living lettuce. C) The packaged clamshells containing living

lettuce with intact roots were transported on ice. D) To prevent contamination

of leaves by splashing, a Ziploc® plastic bag was used to encase the head with

only a small hole to allow roots to protrude. E) The roots soaking in the

x

nutrient solution containing Salmonella for 10 minutes. F) Wrapped roots

forming a knot. G) The knot formed. H) Immediately transfer the

lettuce with wrapped roots to clamshell container. I) Store the packaged

lettuce in 4˚C or 12˚C, respectively, to be processed on sampling

days………………………………………………………………………………...…….86

Figure 4.2. Enumeration of Salmonella recovered from lettuce roots and leaves

of living lettuce held at 12˚C throughout the 18-day shelf life. Bars reflect the

average numbers of log CFU/g from 3 replicates destructively processed per

sampling day recovered by plating on XLT4-Rif and incubated at 37˚C for 48 h.

Each error bar is constructed using 1 standard deviation from the mean. Samples

not connected by same letter are significantly different (P<0.05)……………………….87

Figure 4.3: Enumeration of Salmonella recovered from living lettuce roots and

leaves held at 4˚C throughout the 18-day shelf life. Bars reflect the average

numbers of log CFU/g from 3 replicates destructively processed per sampling day

recovered by plating on XLT4-Rif and incubated at 37˚C for 48 h. Each error bar is

constructed using 1 standard deviation from the mean. Samples not connected by same

letter are significantly different (P<0.05)…………..…………………….………..…….88

1

CHAPTER 1: INTRODUCTION AND JUSTIFICATION

For thousands of years, food safety has been a concern for consumers simply because

food is recognized as a potential carrier for harmful bacteria. Food-borne illnesses present a

serious public health burden worldwide. The overall burden of food-borne illness in the United

States is estimated at 48 million cases per year and estimated costs of food-borne illnesses are

$152 billion per year (9). Ingestion of food contaminated by bacteria, parasites or viruses results

in illnesses for one in six Americans each year (3). Outbreaks, defined as two or more people

with the same symptoms and identical etiological agent, are increasingly attributed to fresh

produce. Outbreaks attributed to consumption of fresh produce have increased from <1%

(13/1,857 outbreaks) in the 1970s to 6% (114/1,788 outbreaks) in the 1990s (10). The frequency

of produce outbreaks increased from two outbreaks per year in the 1970s to 16 per year in the

1990s (10).

Food-borne outbreaks associated with leafy greens have increased by 38.6% (6,7).

“Leafy greens” refers to vegetables including lettuce, cabbage, endive, escarole, spinach,

broccoli, collard greens, turnip greens, mustard greens and kale (12). Leafy greens are

recognized as a vehicle for transmission of pathogens that have the ability to cause food-borne

illness outbreaks (1, 2, 4, 6, 7). Between 1973-2006, a total of 502 food-borne outbreaks

associated with leafy greens were reported in the United States, and 35 outbreaks were attributed

to serotypes of Salmonella enterica (6,7).

Pre-cut, pre-washed and packaged leafy greens that require little preparation are

increasing in demand by consumers (1). Since the 1960s, per capita consumption of different

types of leafy greens have increased from 21.4 pounds per capita to a record high in 2004, with a

2

total lettuce consumption of 33.2 pounds per capita (13). Lettuce is a leading food crop in the

United States with an annual profit of more than 2 billion dollars in year (14).

While the bulk of lettuce and leafy greens sold in the United States are field grown,

hydroponic production in greenhouses is increasing in popularity. Much of the lettuce produced

hydroponically is packaged with intact roots in a plastic clamshell and marketed as “Living

Lettuce”. The survival and behavior of human pathogens within hydroponic lettuce production

systems is not well understood. It is known that post-harvest activities such as washing,

handling, and packing can increase the amount of contaminated product through cross-

contamination ( 5, 6, 8, 9, 11). The objectives of this study were to (i) Quantify the transfer of

Salmonella enterica serotype Enteritidis from inoculated roots to the leaves of mature Butterhead

lettuce packaged as “living lettuce” in a clamshell with intact roots, (ii) Determine the survival of

S. Enteritidis on roots and edible tissue of living lettuce stored at 4˚C and 12˚C throughout a

typical product shelf life, (iii) To investigate the effectiveness of the current sampling strategies

for quantification of S. Enteritidis on living lettuce. We hypothesize that post-harvest survival of

Salmonella enterica from inoculated roots to the leaves can persist at FDA recommended storage

temperatures and grow at temperature abusive conditions. We also hypothesize that sampling

strategies can be improved to increase detection of Salmonella on leafy greens.

These objectives will provide important information about living lettuce and Salmonella

enterica serotype Enteritidis during post-harvest handling. The techniques described here can be

used to increase awareness of maintaining safe handling practices of living lettuce in hydroponic

systems and guide risk management strategies. Therefore, understanding current harvesting,

transport, and handling practices is important to identify potential risks and implement strategies

to reduce cross-contamination. Control measures need to be identified to reduce risks and

3

improve the safety of leafy greens. These insights will be valuable in designing guidelines

targeting post-harvest handling practices in commercial-scale, hydroponic production of leafy

greens. Application of GAPs (Good Agricultural Practices) and GHPs (Good Handling

Practices) are suggested to increase food safety of leafy greens by minimizing pathogen

contamination during post-harvest handling.

4

REFERENCES

1. Beuchat, L. R. 1996. Pathogenic microorganisms associated with fresh produce. Journal

of Food Protection. 59:204-216.

2. Brecht, J. K. 1993. Physiology of lightly processed fruits and vegetables. HortScience.

28:472.

3. Centers of Disease Control and Prevention. 2011. CDC estimates of food-borne illness in

the United States. Available at:

http://www.cdc.gov/foodborneburden/PDFs/FACTSHEET_A_FINDINGS_updated4-13.pdf.

Accessed July 2012.

4. Centers of Disease Control and Prevention. 1998. Outbreak of Campylobacter enteritis

associated with cross-contamination of food-Oklahoma, 1996. Available at:

http://www.cdc.gov/mmwr/preview/mmwrhtml/00051427.htm. Accessed July 2012.

5. Davis, H., J. P. Taylor, J. N. Perdue, G. N. Stelma, R. Rowntree, and K. D. Greene. 1988.

A Shigellosis outbreak traced to commercially distributed shredded lettuce. American Journal of

Epidemiology. 128:1312-1321.

6. Herman, K. M., T.L. Ayers, and M. Lynch. 2008. Foodborne disease outbreaks

associated with leafy greens 1973-2006. International Conference on Emergining Infectious

Diseases. Location and page numbers

7. Lynch, M., R. V. Tauxe, and C. W. Hedberg. 2009 The growing burden of foodborne

outbreaks due to contaminated fresh produce: risks and opportunities. Epidemiology and

Infection:volume?307-315.

8. Moore, C. M., B. W. Sheldon, and L. A. Jaykus. 2003. Transfer of Salmonella and

Campylobacter from stainless steel to romaine lettuce. Journal of Food Protection. 66:2231-

2236.

9. Scharff, R. L. 2010. Health-related costs from food-borne illness in the United States.

The Produce Safety Project At Georgetown University. www.producesafetyproject.org.

Accessed July 2012.

10. Sivapalasingam, S., C. R. Friedman, L. Cohen, R.V. Tauxe. 2004. Fresh produce: a

growing cause of outbreaks of foodborne illness in the United States, 1973 through 1997.

Journal of Food Protection. 67:2342-2353.

11. Stafford, R. J., McCall, B.J., Neill, A.S., Leon, D.S., Dorricott, G.J., Towner, C.D. and

Micalizz, G.R. 2002. A statewide outbreak of Salmonella Bovismorbificans phage type 32

infection in Queenland. Communicable Diseases Intelligence Quartely Report. 26:568-573.

5

12. U.S. Department of Agruiculture. Economic Research Service. 1998. Leafy greens:

foundation of the vegetable industry. Available at:

http://webarchives.cdlib.org/sw1s17tt5t/http:/ers.usda.gov/publications/agoutlook/jan1998/ao248

b.pdf. Accessed July 2012.

13. U.S. Department of Agruiculture. Economic Research Service. 1960-2010. U.S. lettuce

per capita. Available at:

http://usda.mannlib.cornell.edu/MannUsda/viewDocumentInfo.do?documentID=1576. Accessed

July 2012.

14. U.S. Department of Agruiculture. Economic Research Service. 2009. U.S. lettuce

production value. Available at:

http://usda.mannlib.cornell.edu/MannUsda/viewDocumentInfo.do?documentID=1212. Accessed

July 2012.

6

CHAPTER 2: LITERATURE REVIEW

Globalization of the food supply has made fruits and vegetables available regardless of

season. Improvements in production yields, distribution, and trade policy allow perishable foods

to travel long distances with minimal spoilage (12). Consumers are more aware of the important

nutrients and health benefits associated with eating fresh produce. Fruits and vegetables have

fundamental nutrients that offer key dietary advantages such as essential minerals, vitamins,

antioxidants and other substances that can reduce or prevent diseases with the ultimate goal of

promoting a healthy lifestyle (4). High quality produce subjected to minimal processing is in

demand by consumers who are often willing to pay more for produce grown locally with

sustainable methods.

Fresh produce-related illnesses monitored by Centers for Disease Control and Prevention

surveillances (CDC) have been increasing from <1% in the 1970s to 6% in the 1990s and

continues to rise (73). The number of produce-related outbreaks increased from two outbreaks

per year in the 1970s to 16 per year in the 1990s (73). The sharp rise in produce-related

outbreaks is an ever-increasing challenge for the food industry. A Produce Safety Project report

from March 2010 estimated the annual economic burden of food-borne illness in the United

States to be $152 billion, of which $39 billion was from economic losses associated with

contaminated domestic produce (68). CDC states that reducing food-borne disease by 10 percent

would prevent 5 million people infected annually (21). Widespread produce outbreaks have

been documented including: Salmonella Braenderup infections from mangoes, Escherichia

coli O157:H7 linked to spinach and romaine lettuce, Salmonella Newport associated with alfalfa

sprouts, Salmonella Litchfield from cantaloupes, and Salmonella Typhimurium linked to

tomatoes (24).

7

Leafy greens are traditionally perceived as a relatively safe product, and it is difficult to

convince consumers that such nutritious greens can be highly contaminated with pathogens.

Leafy greens are recognized as a vehicle for transmission of pathogens that have the ability to

cause food-borne illness outbreaks (22, 23, 24, 25).

Hydroponic leafy greens production has been increasing in popularity and the importance

of contamination prevention must be emphasized at all stages of production. Prevention of

contamination is vital to leafy greens safety and to maintain the economic value of the

commodity. Further efforts are necessary to understand the preferred routes of contamination and

the appropriate intervention to reduce the impact of food-borne outbreaks. Assessing continuing

reports of food-borne disease outbreaks is critical towards the success of current and future

prevention strategies. These insights will be valuable for developing safe hydroponic practices to

improve pre- and post-harvest safety of leafy greens, particularly living lettuce.

Production Value, Per Capita Consumption, and Exported and Importation of Fresh

Lettuce

According to the Economic Research Service (ERS) and United States Department of

Agricultural (USDA) databases, the convenience of minimal processing such as pre-cut and pre-

washed lettuce that requires little preparation is increasing in popularly (81, 83, 85, 86). It was

reported that the per capita consumption of all lettuce varieties has been increasing since 1960

with 21.4 pounds per capita and in 2004 total lettuce consumption reached a record high of 33.2

pounds per capita (85). Since 2004, the per capita consumption has dropped slightly and in 2010

28.2 pounds per capita of lettuce was consumed (85). In terms of production value, lettuce is a

leading vegetable crop in the United States with an annual profit of more than 2 billion dollars

per year (86).

8

The majority of the US lettuce crop is consumed in country while some is also exported.

The US exports lettuce to selected countries such as Mexico and Canada. In 2009, Mexico

imported a net worth of $88,438,355 and 223,788,972 pounds while Canada imported a net

worth of $25,106,690 and 66,609,395 pounds of all various lettuce (77).

Statistical Analysis of Food-borne Illnesses Outbreaks Burden in the United States

Food-borne illnesses present a serious public health burden in the United States.

It is estimated that 48 million people will suffered from food-borne illness annually in the United

States (21). Multiple recognized surveillance databases were utilized to estimate the overall

food-borne illnesses burden in the United States. Data analysis was collected between 2000 and

2008 and based on US population in 2006 (299 million persons), food-borne illnesses were

associated with one of the 31 known specific pathogens (67).

Table 2.1: The number of people affected by food-borne illnesses, hospitalizations, and deaths

linked to 31 known pathogens in 2000-2008.

Overall estimate of 31 known pathogen contributors of Food-borne Diseases, 2000-2008 (67).

Food-borne illnesses 9.4,000,000

Hospitalizations 55,961

Deaths 1,351

The Food-borne Disease Outbreak Surveillance System survey concluded that between

2003-2007 the number of produce-related food-borne illnesses in the U.S. was 19,677,547 and

that Virginia ranked number 12 in number of reported cases (500,395). California ranked

number 1, contributing to 2,372,499 cases of produce-related food-borne illnesses (68).

Norovirus and serotypes of non-typhoidal Salmonella enterica are the leading pathogens

accountable for causing food-borne illnesses in the United States (21). Estimated annual number

9

of Norovirus illness was 5,461,731 in 2011 and non-typhoidal serotypes of Salmonella were

1,027,561 cases in 2011. Non-typhoidal serotypes of Salmonella and Toxoplasma gondii are

food-borne pathogens that cause the largest number of deaths annually, with 378 and 327 cases,

respectively (21). In a 2011 journal, Ranking the Risks: The 10 Pathogen-Food Combination

with the Greatest Burden on Public Health, poultry was rated first with a liability cost of

$2,462,000 and 180 associated deaths recorded in the United States. Fresh produce was listed as

fourth, accounting for a $1,405,000 cost of illness and 134 deaths (9).

Produce-associated contamination between 1973 and 1997 accounted for 190 outbreaks,

16,058 illnesses, 598 hospitalizations, and 8 deaths (73).

Table 2.2: The number of food-borne outbreaks, illnesses, and deaths cases associated with

leafy greens reported from 1973-2006.

Food-borne Outbreaks Associated with Leafy Greens Reported From 1973-2006 (67,68)

Outbreaks 502 (4.8%)

Illnesses 18,242 (6.5%)

Deaths 15 (4.0%)

Salmonella Outbreaks 35 (10.4%)

Notable Outbreaks Associated with Leafy Greens

Food-borne illness outbreaks can have a severe economic impact on the food industry.

Salmonella enterica, a common food-borne disease, is a problem recognized internationally. In

2004, multiple illnesses have been linked with imported ‘Rucola’ lettuce (Eruca sativa-known as

rocket salad or arugula in the US) and mixed salad blends in several European countries (61).

Three isolates of Salmonella Thompson were identified from arugula and confirmed as the

outbreak strain by pulsed-field gel electrophoresis by the Norwegian Institute of Public Health

10

(NIPH) on November 15, 2004. The imported arugula was traced to an Italian producer and the

contamination source was determined to be irrigation with non-potable water. This case

emphasized the importance of using good water quality for leafy green production.

In the United States, contaminated minimally processed bagged spinach was linked to a

multistate outbreak of food-borne, Escherichia coli O157:H7 (22). The 199 people affected and

3 deaths were recorded. Of those infected, fifty-one percent were hospitalized and sixteen

percent developed kidney failure due to hemolytic-uremic syndrome (HUS). The outbreak

affected 26 states with Nebraska having the highest number of confirmed cases with 11. The

FDA traced the outbreak to Natural Selection Foods LLC of San Juan Bautista, California and

the spinach was likely contaminated by run-off from adjacent dairy pastures (22).

A December 2011, multistate outbreak infected 60 people was linked to romaine lettuce

contaminated by a strain of E.coli O157:H7 (23). The outbreak was confirmed in 10 states:

Arizona, Arkansas, Georgia, Illinois, Indiana, Kansas, Kentucky, Minnesota, Missouri, and

Nebraska. There were 37 cases in Missouri the largest of any state. Amongst those who were

infected, 30 were hospitalized and 2 were diagnosed with hemolytic uremic syndrome (HUS).

An investigation was conducted to determine the original source of the outbreak in order to

contain and prevent it from spreading. Evidence concluded that the romaine lettuce was served

on salad bars at all locations of grocery store Chain A. The romaine lettuce came from a single

lettuce processing facility distributor, which suggests that the romaine lettuce was contaminated

during transportation prior to its arrival of the designated grocery store Chain A (23).

Salmonella spp. outbreaks associated with lettuce have not been documented recently,

however a number of recalls based on presence of pathogen have occurred, indicating there is

still a potential risk of food-borne illness.

11

Table 2.3: Recalls reported from 2009-2012 associated Salmonella enterica with various salad

blends from field production (78).

Brand name Date of

recall

Microorganism

Identify Product Reason

Plan of

Action Location

Fresh

Express

October

11,

2012

Salmonella

Hearts of

Romaine

Salad

A random

sample

revealed a

positive

result for

Salmonella

Voluntary

recall

North

Carolina

Pacific

International

Marketing

July 6,

2012 Salmonella

Bulk

Romaine

Lettuce

Salmonella

tested

positive

taken at the

field

production

Voluntary

recall

California,

Nevada

Dole Fresh

Vegetables

April

14,

2012

Salmonella

Seven

Lettuce

Salad

A random

sample

revealed a

positive

result for

Salmonella

Voluntary

recall Multistate

Taylor Farms

Retail

October

19,

2011

Salmonella

Various

salad

blends

A random

sample

revealed a

positive

result for

Salmonella

Voluntary

recall Multistate

Thorntons,

Inc.

October

1, 2011 Salmonella

Garden

and chef

salads

Potential

cross-

contaminati

on of grape

tomato

linked

Salmonella

Voluntary

recall Multistate

J&D Produce

Decem

ber 28,

2010

Salmonella Fresh

greens

A positive

test linked

to

Salmonella

on curly

parsley

Voluntary

recall Multistate

Fresh

Express

May

24,

2010

Salmonella

Various

Romaine

Ready-

to-eat

A random

sample

revealed a

positive

Voluntary

recall Multistate

12

salad

blends

result for

Salmonella

Tanimura &

Antle

July

21,200

9

Salmonella Romaine

Lettuce

A random

sample

revealed a

positive

result for

Salmonella

Voluntary

recall Multistate

On April 14, 2012, Dole Fresh Vegetables voluntarily recalled 756 cases of DOLE®

Seven Lettuces salad distributed in fifteen U.S. states (80). A Dole lettuce product tested positive

for Salmonella enterica during a random collection sample test in New York. The source of

contamination was unknown. No illnesses were reported; however, if precautionary actions had

not been taken, severe consequences leading to food-borne illnesses outbreak could have

occurred (80).

To the best of our knowledge there have been no attributed outbreaks of Salmonella spp.

associated with lettuce produced hydroponically. However, Salmonella outbreaks of alfalfa

sprouts grown hydroponically have been reported (79). Other recalls traced back to 4 oz Alfalfa

Sprout Cups produced by Arizona Hydroponic Farming LLC of Eloy, Arizona. No illnesses were

reported during this episode; however, bacterial contamination of sprouts is well documented.

This establishes that contamination occurs in hydroponic environments, and that outbreaks of

food-borne illness are possible from hydroponically produced lettuce (79).

Characteristics and Morphology of Salmonella enterica

Salmonella is characterized as a motile, non-spore forming, gram-negative, rod-shaped

bacterium in the family of Enterobacteriaceae (37). Salmonella is categorized into two species,

Salmonella enterica and Salmonella bongori. Salmonella enterica is arranged into 6 subspecies

(37). Salmonella enterica is further sub-divided into serovars. The Kaufmann-White typing

13

scheme classifies over 2,579 serovars, based on their reactivity to surface LPS and flagella

antigens (37).

The majority of the serovars in Salmonella enterica subspecies enterica are classified as

non-typhoidal Salmonella because they result in symptoms associated with gastrointestinal

illness. However, a few serotypes, the most notable being S. Typhi, are associated with much

more dangerous systemic infections referred to as Typhoid fever (37). With the exception of S.

Typhi, Salmonella enterica is normally found in the intestinal tracts of reptiles, animals, and

birds and is transmitted to humans when feces contaminate food or water (12).

Young and/or elderly with weak immune systems, or who are immuno-compromised due

to chronic illness, HIV or chemotherapy for cancer, are at a higher risk of acquiring Salmonella

enterica infections (37). Fewer than 100 cells of Salmonella enterica may be sufficient to cause

disease in humans, though the infectious dose is dependent on age, immunization status, and

epidemiology of various serotypes of Salmonella, which vary in their virulence for humans (14).

Salmonella enterica can pose serious public health concerns, particularly in the produce

industry, because it has the ability to persist at low temperatures (18). Salmonella enterica has

been associated with outbreaks of human disease from commercial scale field and greenhouse

produce production.

FoodNet analyzed trends of food-borne diseases between 1996-2010 and results show

Salmonella enterica infections have not declined in 15 years, and steadily increased between

2006-2008, increasing public health concerns (26). Different types of Salmonella genotypes and

a variety of reservoir contamination are some of the major challenges to reducing the numbers of

Salmonella infections that occur in a wide variety of foods (26).

14

Pre-Harvest Handling and Potential Reservoirs of Contamination

Leafy greens can be contaminated at any point during growth, transport, or handling.

Reducing pre-harvest contamination is an important line of defense in decreasing numbers of

produce-associated food-borne illnesses. Optimizing pre-harvest treatments can improve the

microbiological safety of leafy greens. Prior studies have extensively demonstrated the

transmission of bacteria to produce in a variety of ways including animal feces, soil

contamination, improperly composted manure, sewage, runoff from pastureland, storm related

events such as floods, and contaminated irrigation water (16,29,59,71). It is important to

establish and maintain good human hygiene practices to reduce leafy greens-associated

outbreaks.

The use of manure and other fertilizer materials derived from animal waste, water quality

for irrigation and washing, and the hygiene of workers and facilities are areas of emphasis to

reduce pre-harvest contamination (12). Gallegos-Robles et al. (34) assessed several cantaloupe

farms and tested 28 samples of cantaloupe. Of those samples, 43% (12 of 28) tested positive for

Salmonella enterica, which can survive in soil, irrigation water sources, and on the hands of

workers who handle cantaloupes (34).

Ponds, rivers, streams, municipal water, and reclaimed water are common sources of

irrigation water used for produce production (74). Irrigation water can introduce pathogens from

animal fecal matter directly or indirectly on production fields. The contaminated feces can be

activated by water and moved through irrigation systems (5,53).

A 2006 outbreak of Escherichia coli O157:H7 in bagged spinach was traced back to a

California production site (43). The outbreak strain matched isolates from feral swine, cattle

feces, surface water, sediment and soil approximately one mile from the spinach production

15

fields. Livestock carriers of Escherichia coli O157:H7 feces served as vehicles of transmission

to water and soil contamination (43). Cooley et al. (27) conducted an assessment of Escherichia

coli O157:H7 for 19 months in a Salinas Valley, California watershed characterized by surface

water, creeks, and streams.

Fifteen of 22 water sources in the watersheds sampled tested positive for Escherichia coli

O157:H7. Watersheds linked with Escherichia coli O157:H7 frequently contained cattle. In

addition, heavy rain or flooding events caused an increased flow rate that contributed to higher

incidences of Escherichia coli O157:H7 (27).

Irrigation methods can impact the degree of microbial contamination and its survival on

leafy greens. Solomon et al. (71) showed that a greater number of lettuce samples tested positive

for E. coli O157:H7 when applied through spray irrigation compared to drip irrigation. This was

a particular concern where water sources were limited and reclaimed recycled water was an

attractive alternative method of irrigation water (71). Routine sampling and maintaining potable

water sources was highly recommended to reduce food-borne associated outbreaks (76). This

study reinforces the importance of choosing premium quality irrigation water and knowing its

origin and distribution.

Prior studies have demonstrated the persistence of Salmonella Typhimurium on leafy

greens in the fields treated with contaminated composted manure and irrigation water (51, 70).

Lapidotand et al. (51) revealed that Salmonella Typhimurium has the ability to persist in

favorable environmental conditions for 161 days in the manure composts applied as fertilizer for

commercial lettuce production. A maximum amount of time should be allowed between the final

application of properly composted manure and harvest, to kill pathogenic bacteria in the field,

reducing the risk contamination (41).

16

Pre-harvest prevention strategies are often implemented to maintain disease control.

Suggested strategies such as fencing the production fields to provide barriers to domestic and

wild animals, selection and placement of production fields to be away from high traffic of

domestic and wild animals, and ensuring the production fields have enough distance from

irrigation water sources to prevent contamination. It is important to establish clear and marked

barriers to prevent cross-contamination, and reducing human activities may prevent the

distribution of food-borne disease.

Post -Harvest Packaging and Handling and Potential Reservoirs of Contamination

Post-harvest causes of contamination may include one or more of the following: poor

worker hygiene, improper sanitation of equipment, transport containers and transportation

systems, cross-contamination, wild and domestic animals, insects, irrigation water, ice,

temperature abuse during storage, packaging, or preparation (12, 19). Transportation systems

such as trucks that were previously used to transport animals or meats and then improperly

sanitized could cross-contaminate produce during transport from farms to distributors (73). The

economic viability of the food industry and consumer safety relies heavily on prevention of

contamination (68).

After harvest, further processing of leafy greens such as slicing, shredding, squeezing and

peeling, can provide opportunities for contamination to occur via the infected hands of the

handlers, contaminated water for final washing or rinsing, cross-contamination from one item to

another or equipment. Water is a recognized vehicle that allows pathogens to be distributed from

one area to another. During post-harvest processing, clean water baths used to wash and rinse

produce are vital to reduce cross-contamination. The contaminated water could promote cross

contamination across commodities in the same facility (73). Wachtel et al. (88) concluded that

17

adding one inoculated lettuce leaf to 350 g of chopped dry lettuce resulted in 100%

contamination of the lettuce leaves, testing positive for E. coli O157:H7 even when stored in

water at temperatures of 4˚C and 25˚C for 20 hours. E. coli can continue to increase in numbers

due to the release of nutrients from produce at storage temperatures above 8˚C (88). The water

availability, temperature, tissue damage, nutrients, and native microbiota are all factors that can

sustain pathogens associated with produce (62). The risks for amplification of pre-existing

pathogens on fresh produce include release of nutrients due to mechanical injury and other post-

harvest handling.

Cross-contamination is a recognized source of transferring pathogens to leafy greens that

potentially could lead to food-borne outbreaks. Contamination may be present through

inadequate hygiene, both of workers hands and of processing facilities and equipment.

Emphasizing the importance of hand washing and maintaining clean processing facilities is

critical to reduce the risk of food-borne outbreaks. For example, 41 cases of Salmonella

Bovismorbificans were linked to cross contamination from an inadequately sanitized cutting

wheel used to shred multiple heads of lettuce (72). In another shredded lettuce-associated

outbreak, the outbreak strain of Shigella was isolated from an infected worker and found in the

processing plant (28). Moore et al. (56) showed that serotypes of Salmonella and Campylobacter

jejuni can both be transferred from contaminated stainless steel surfaces to wet or dry lettuce

even when the surface contamination occurred 2 hours previously (56). In Oklahoma, an

outbreak of Campylobacteriosis was traced back to cross-contamination of lettuce by

contaminated utensils and hands used to process raw chicken (25).

18

Molecular Mechanisms of Attachment of Salmonella spp. to Plants

Understanding the preferred mechanisms of attachment of pathogens, particularly

Salmonella enterica, will enhance intervention strategies to prevent outbreaks associated with

leafy greens. Food-borne pathogens have the capacity to thrive outside their hosts by adjusting

to their new surroundings. Variation among cultivars, different genotypes, physiological state

and type of fruit or vegetable, can influence the microbial communities associated with produce.

Klerks et al. (46) proposed that Salmonella enterica serovars interact differently with different

lettuce cultivars, which greatly influence their survival. Various ecological niches of pathogens

can vary depending on surface morphology, metabolic behavior, and tissue arrangement of the

leafy greens (11, 52). Brandl et al. (16) analyzed the effects of leaf age as a risk factor for

distribution of E.coli O157:H7 and Salmonella associated with lettuce during pre- and post-

harvest stages. The study showed both pathogen populations increased 16 to 100 fold on young

leaves compared to older leaves, respectively, when stored at 28˚C. The analysis of exudates

collected from leaf surfaces of different ages showed that young leaves were 2.9x richer in total

nitrogen and 1.5x richer in carbon compared to the older leaves (15). In contrast, Kroupitski et

al. (49) demonstrated that older lettuce leaves were the preferred choice of attachment of

Salmonella Typhimurium compared to younger leaves. It was suggested that the degree of

attachment correlated to the lettuce surface morphology, which also changed with leaf

development. In addition, it was observed the preferred attachments on surfaces were closer to

the petiole, 7.7 log CFU/g, and on the lower surface of the leaf (49).

Cellulose, fimbriae, and O-antigen capsule are important non-specific bonding

attachment mechanisms of food-borne pathogens to leaf tissue surfaces (6, 7, 18). Non-specific

attachment to plant surfaces in Salmonella enterica is encoded by genes including yihO, bcsA,

19

rpoS, and agfD (6, 7). The gene rpoS is recognized as a general stress regulator for Salmonella

enterica. It promotes environmental survival and is important for biofilm formation, a strategy

that may promote survival on leaf surfaces. Other biofilm formation genes are also important for

plant colonization including bcsA, which triggers the production of cellulose, yihO, which

activates the glucuronide transporter required for capsule transport to the cell surface, and gene

agfD, which regulates the production of cellulose and O-antigen capsule contributing to

attachment (6, 7). Kroupitski et al. (50) demonstrated a polystyrene plate model to evaluate

biofilm production of various Salmonella enterica serovars on intact and cut lettuce leaves, and

discovered strong biofilm producers attach better than weak biofilm producers. Patel et al. (63)

showed that Salmonella Tennessee and Thompson generated stronger biofilm formation on

lettuce leaf and cabbage leaf surfaces compared to Salmonella Newport, Negev or Braenderup,

suggesting that attachment can be influenced by serovar characteristics. Overall, the results

showed Salmonella enterica preferred attachment to lettuce rather than cabbage at intact and cut

surfaces (63).

Survival and growth of human pathogens can also be influenced by the presence of other

microorganisms. Wells et al. (90) observed a positive correlation between leafy greens infected

with bacterial soft rot pathogens and those that were also positive for presence of human enteric

pathogens, particularly Salmonella enterica. Bacterial soft rot is a post-harvest plant disease

associated with poor handling, storage abuse, or poor sanitation. Results revealed that out of 401

samples affected by bacterial soft rot, 66% tested positive for Salmonella. In comparison only,

30% of 402 healthy samples tested positive, indicating a likely association between bacterial soft

rot and Salmonella. Post-harvest plant disease such as rotten tissue can provide nutrients that

would be available to bacterial microorganisms helping them to grow and persist (90).

20

Previous studies have proposed that Salmonella enterica serovars can colonize the

rhizosphere, depending on cultivar types, produce types, different genotypes and physiological

state of the produce influenced the route of preferred colonization mechanisms (7, 10, 11, 45-47).

Sugar sources, such as fructose, in the root exudates attract Salmonella making colonization of

the rhizosphere more likely (10, 47). A better understanding of attachment mechanisms used by

pathogens, particularly Salmonella enterica, will improve food safety by helping to implement

intervention strategies to reduce bacterial contamination of leafy greens during hydroponic

production.

Food Safety and Prevention Strategies

Proactive food safety should include preventative measures with a reduction in food-

borne illness outbreaks as the ultimate goal. Leafy greens sanitation practices should start at the

beginning of the production cycle with seed germination and continue through subsequent

production stages such as growing, harvesting, post-harvest handling, transportation, and

processing and preparation.

GMPs (Good Manufacturing Practices), GAPs (Good Agricultural Practices, and HACCP

(Hazard Analysis and Critical Control Point) are critical and fundamental preventative methods

to ensure the microbiological safety of produce by limiting contamination by human pathogens.

HACCP focuses on food safety and implements preventive controls that can eliminate or

minimize food safety hazard risks posed by microbiological, chemical, and physical factors (8).

Producers and management should consider incorporating a HACCP plan for all post-harvest

handling processes, including those in the greenhouse and packing house. Currently, the US

Food and Drug Administration, FDA, does not require producers to implement the HACCP

systems for leafy greens that are required for other food commodities.

21

A joint effort by the United States Department of Agriculture (USDA) and the US Food

and Drug Administration (FDA) developed voluntary guidelines entitled “Guide to minimize

microbial food safety hazards for fresh fruits and vegetables”. This document highlights good

agricultural practices (GAPs) to reduce field contamination and good management practices

(GMPs) to minimize the risk of microbial contamination in fresh leafy greens (83). Specific

GAPs guidance has been developed for field-grown lettuce and leafy greens, yet implementation

of these practices can only reduce but not completely eliminate microbiological contamination

associated with fresh leafy vegetables (81). At this time there are no guidelines or practices

required by law regarding post-harvest handling of leafy greens grown hydroponically.

Cooperation by U.S. government and producers is strongly encouraged to focus on the

improvement of the microbiological quality of leafy greens in hydroponic production. These

united efforts to establish practices and risk management practices for hydroponic production are

vital to reducing the impact of food-borne illness outbreaks. These actions will ensure consumers

a safer supply of leafy greens, which improve public health. Continuation of research on post-

harvest contamination by human pathogens and their mechanisms of survival in field and/or

hydroponic production setting are needed to develop food safety intervention strategies

throughout critical stages of leafy greens production.

In addition to prevention, reducing the spread of outbreaks and educational programs on

biological contamination are important factors for maintaining food safety. Educating workers of

potential risks and prevention strategies can greatly reduce contamination caused by food-borne

pathogens. It is critical that workers in the leafy greens industry and consumers understand the

basic principles of food safety when handling raw leafy greens. In addition to proper training,

monitoring, and preventative action are needed to maintain food safety practices (3).

22

Methods of Leafy Greens Sanitation

Proper sanitation methods are an important intervention steps at critical points during

production, harvest, or preparation. Using potable water to wash leafy greens can remove loose

surface contaminants such as dirt and insects, ultimately lowering the bacterial load by 1 to 2 log

CFU/g (49). The reduction in bacterial load can help both extend shelf life and improve quality

of leafy greens, but does not completely remove human pathogens from contaminated leafy

greens (66).

The Food Safety and Inspection Service (FSIS), recommends all water used for post-

harvest washing of produce consist of potable water plus chlorine 50 to 200 parts per million of

free (available) chlorine at a pH of 6.8 to 7.2 with a contact time of 1-2 minutes to limit cross-

contamination during washing and between lots (13). To optimize sanitation, it is important to

monitor the pH of the wash water, the concentration of free chlorine, the concentration of

inorganic and organic matter, and the temperature of the solution (13). Poor sanitation may lead

to bacterial contamination of washing systems. For example, wash water used in the preparation

of pre-washed bagged lettuce can transfer bacteria from one batch to the next, with the potential

of infecting a full day’s production of lettuce.

Sanitizer agents reduce, but do not completely eliminate, microorganisms established on

food surfaces, making their removal during processing and handling procedures difficult.

Bacteria can infiltrate cracks in leaves where they avoid contact with chemical sanitizing agents

ineffective. Furthermore, the bacteria can form biofilms with strong attachments to the leaf that

prevent surface removal (18, 31, 66). Weissinger et al. (89) applied inoculated Salmonella

Baildon to shredded lettuce to determine the efficiency of chlorine sanitation on bacteria during

storage at 4˚C for up to 12 days. Shredded lettuce was inoculated with 3.60 log CFU/g of S.

23

Baildon and immersed into a 200 mg/mL free chlorine solution for 40 seconds, which resulted in

less than 1 log reduction, suggesting this treatment was ineffective for eliminating the pathogen

(89). Zhang et al. (91) reported that the most effective surface disinfection methods for

inactivating Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes from lettuce

leaves and roots was dipping in 80% ethanol for 10 seconds followed by immersion in 0.1%

HgCl2 for 10 minutes. However, these treatments are much too harsh and use compounds that are

dangerous for human consumption (91). New strategies to reduce spoilage and to inactivate

pathogens using ozone (12), gamma irradiation (12), and hydrogen peroxide treatments have

been effective but are not currently used in the industry (91).

Hydroponics

Hydroponics technology has increased in popularity for greenhouse production of

vegetables, particularly leafy greens. In Virginia, Red Sun Farms will build hydroponic

productions on 45 acres in the New River Valley Commerce Park and hope to create 205 jobs

over five years (30). Reports revealed hydroponic production generated $544 million in revenue

and expected to have an annual growth of 7.8% in US (40). Hydroponics is a production method

used for growing plants in plant-nutrient supplemented water without soil (36). This alternative

agricultural practice has the potential to reduce land requirements for vegetable production by at

least 75% and to reduce single use irrigation water by 90%, appealing to producers with limited

land space and access to water resources (15). Recaptured and recycled drainage water yielded a

33% reduction in water production of cucumbers without reducing yield. This creates an

attractive solution of using recaptured recycled water to reduce environmental impacts of

drainage water discharges, thus reducing pollution issues. Using recycled water can be

appealing to producers by saving revenue and becoming more eco-friendly. This eco-friendly

24

strategy reduces water and sewage costs for aquaculture producers while producing a

horticultural crop with its own economic value (36). However, despite some of the attractive

features, a major downside include the potential for roots to be contaminated as roots are

constantly soaked in recirculating water system. This could increase potential risk of root access

by providing nutrients to harmful human pathogens including 17 species of Phytophthora, 26 of

Pythium, 27 genera of fungi, 8 species of bacteria, 10 viruses, and 13 species of plant parasitic

nematodes (39,55, 65,75).

Finding a reliable source of fresh water is challenging for conventional producers

worldwide. The limited water resources are costly to sustain production, thus increasing the use

of low quality water for irrigation on production fields is attractive. However, surface water may

raise the risk potential of food-borne infections (33,74). The U.S. Environmental Protection

Agency (EPA) conducted a survey of water quality standards in 2004 in the United States.

Analysis indicates 44% of the streams, 64% of the lakes, and 30% of estuaries were below water

quality standards for designated uses such as drinking, swimming or fishing (84). Previous

studies indicate Salmonella enterica can swim small distances and in lab studies can serve as a

carrier for contamination of hydroponic lettuce and spinach with Salmonella (42, 50). This is a

concern and needs to be taken into consideration when evaluating the effectiveness of sanitation

methods while screening for bacterial contamination in water.

Selma et al. (69) compared soil and soilless production systems and their impact on

microbiological and sensory qualities of ‘Red Evasion’ (lollo rosso), ‘Red Oak Leaf’ (Jamai)

and ‘Green Butterhead’ (Daguan) lettuce. It was observed that lettuce cultivated in soilless

systems was of better quality; containing higher amounts of phytochemicals, particularly vitamin

C compared to soil cultivated lettuce. The lettuce grown in a soilless system had lower coliform

25

numbers and fewer spoilage associated lactic acid bacteria compared to soil grown lettuce (69).

One attractive benefit that soilless system cultivation offers is the precise control of plant

nutrients and this could potentially increase the yield of leafy greens. Cash crops that have short

reproductive cycles and generate high yields are practical candidates for soilless system (32, 60).

However, contamination routes of leafy greens in a hydroponic production is not well

understand.

Koseki et al. (48) examined two possible routes of contamination of hydroponically

grown spinach leaves. Salmonella enterica, Listeria monocytogenes, and Escherichia coli

O157:H7 were inoculated into the hydroponic system and directly applied to the leaves. Results

indicated the ratio of contamination was 6.93 higher on the roots compared to the leaves (48).

This suggested the primary route of contamination in the hydroponic system was through the

roots rather than direct application of pathogen contamination onto the surface of the leaves (48).

Once the main routes of contamination on leafy greens are identified, strategies to prevent or

reduce hazards must be implemented at its critical control points throughout the food production

chain. This ensures consumers access to a safe food supply of leafy greens and protects the well

being of the consumers resulting in an overall lower risk of food-borne illness outbreaks.

Hydroponic production of Butterhead Lettuce

Lettuce (Lactuva sativa), a dominant component of salad ingredients for its unique

texture and flavor, belongs in the Asteraceae family (formerly Compositae) (57, 58). Lettuce

matures approximately 40-65 days depending on cultivar type and growth conditions (57, 58).

Lettuce is normally composed of 12 to 20 florets, but as low as 7 or as high as 35 is not unusual

(57,58).

26

Lettuce is a cool season crop with temperatures ranging from 60 -65˚F required for

optimum growth (35,57). High temperatures result in tip burn, poorly developed heads and low

density (35). Lettuce flowers when exposed to combinations of high temperature and long day

length exposure so these conditions must be avoided to successfully grow lettuce. The coastal

valleys of California in particular have ideal conditions for high quality lettuce field production

including consistent cool and dry weather, good soils, and a long growing season. Although

hydroponic production of living lettuce is increasing in popularity, the Salinas Valley central

California coast produces 90% of the nation’s lettuce supply (86).

Insect infestation such as aphids, leafhoppers, and cutworms are serious problems

reducing lettuce quality, and they feed on immature heads of lettuce. In addition to pest

infestation, viruses and diseases are serious problems that reduce the yield and quality of lettuce

production. Some of the well-known viruses and diseases are downy mildew, lettuce mosaic, big

vein virus, and corky root bacteria disease (8, 35, 57). Most of these are controlled by growing

resistant cultivars and purchasing certified seed (8, 35, 57). In field production, ‘Crisphead’

lettuce is extensively grown and as a result consumed because of two distinct characteristics

traits: long distance shipping and long shelf life quality. It has lower nutritional quality compared

to other leafy lettuce such as ‘Butterhead’ (35, 58). ‘Butterhead’ is the dominant type used for

living lettuce packaging. The yield of lettuce is the product of head weight and the number of

leaves harvested per unit area, which is important for producers. Besides cultivar differences,

yield can be influenced by several factors such as plant density, head density, cultural practices,

disease/insect control, and the percentage of plants harvested by the shipper (35, 58).

Lettuce has a shallow root system and receiving a steady supply of water is critical to

optimizing growth and development. Drought stress can lead to leaf tip burn that is caused by

27

calcium deficiency. Too much water irrigation can lead to loose head formation or splitting of

lettuce heads, which results in a reduced storage life and quality (35,58). Having a shallow root

system allows a quicker turnover rate of production compared to field production since nutrients

are constantly supplied and available. However, it is unclear how these unique conditions in

hydroponic systems will influence the survival of human pathogens in the system.

Improvements in post-harvest handling will extend shelf-life quality, maintaining color

and appearance, texture, flavor, and nutritional value of leafy greens.

Post-Harvest Handling of Butterhead Lettuce in a Hydroponic Production

Lettuce is a highly perishable commodity, therefore maintaining proper temperature and

relative humidity ensures best quality during shipping and storage. The shelf life of butterhead

lettuce can be extended to 15-18 days by keeping the roots intact and packaging within a plastic

clamshell (8, 35, 58). The majority of butterhead lettuce is hand-harvested since it is very prone

to bruising and damage, which increases its perishability. Vacuum cooling, an effective method

of removing heat from field grown lettuce optimizes shelf-life by maintaining proper temperature

and relative humidity (8, 35, 58); however, due to the high costs associated with the vacuum-

cooling equipment, post-harvest cooling of greenhouse lettuce is often using forced air-cooling.

GMPs (Good Manufacturing Practices) and GAPs are critical, fundamental, and

preventative methods to ensure microbiological safety of living lettuce in a greenhouse setting.

Currently there are no accepted guidelines of GAPs (Good Agricultural Practices) designed for

hydroponic production of butterhead lettuce. Leafy greens sanitation should start from the early

stages of germination and should continue throughout the production stages. These insights will

be valuable in developing agricultural practices for post-harvest safety of butterhead lettuce. The

following recommended guidelines on harvesting living lettuce are simple procedures but vital to

28

maintaining the leafy greens at its premium conditions in the hopes of maintaining good shelf

life stability: 1) Harvest in the morning and in the cool parts of the day. 2) Pack into cool crates

and protect from direct sunlight. 3) Transport in covered vehicles. 4) Cool storage facilities to

store leafy greens are important to remove heat (57).

Intact lettuce roots systems are sometimes harvested to promote good shelf life stability.

Some, large hydroponic lettuce producers package butterhead with the root system wrapped into

a knot (bundled up) beneath the head and both sealed in a plastic bag or box (57). The bagged or

boxed lettuce is packed into crates/trays to be transported to the market. It is important that the

bagged lettuce is packed carefully to avoid bruises and leaf damaged during traveling (57).

Different post-harvest handling practices vary by the size of the hydroponic lettuce production.

Hydroponic butterhead lettuce grown for a local farmers market in Blacksburg, Virginia

as living lettuce with roots intact and packaged into plastic containers. These pictures were taken

by Jessie Waitt, Blacksburg, Virginia in the Spring of 2012.

1. Different growth stages of Butterhead Lettuce ‘Charles’ from Paramount Seed Company

in a Styrofoam floating raft system. As the lettuce matures, they are transferred to a new

raft with wider spacing to promote growth.

29

2. Roots of a living lettuce in a Styrofoam floating raft system. Notice the even distribution

of growth hole spacing in the raft.

3. Post-harvest handling of living lettuce. Rockwool, a man-made mineral fiber that

supports and promotes root growth for hydroponic production, is removed at the

lettuce/roots interface by hand.

30

4. Dead and excessive roots are trimmed by hand. The remaining living healthy roots are

wrapped into a knot by hand.

5. Living lettuce placed in a clear plastic clamshell ready for sale. Please notice the

depression in the bottom of the container, this maintains root health since the moisture

condenses and drips into the depression. This provides water to the roots, which prolongs

the shelf life of lettuce.

31

Typical Production Practices in Small Hydroponic Greenhouses in Virginia

Amber Vallatton, a Virginia Cooperative Extension Agent, discusses her experiences

with five different hydroponic lettuce producers as they work towards GAPS certification

through an e-mail interview (87). One of the questions I specially asked “What are the step by

step post-harvest handling procedures you have observed used by hydroponic lettuce producers

in Virginia?” Below are the 5 different settings of hydroponic lettuce producers in Virginia.

Example 1: A producer uses a vertical stack hydroponic method and grows roots within a

vermiculite mixture growing media. At harvest, living lettuce is placed directly into a

Rubbermaid™ wheelbarrow then transported to a separate packing area. Workers are expected

to follow good hygiene practices by washing hands before wearing nitrile gloves during handling

and packing. Gloves are changed between harvest and packing areas and replaced frequently,

particularly when they become soiled. Knives are washed with soap and water, soaked in

oxidate, aboard spectrum bactericide/fungicide, following label instructions (3/4 oz). Roots

were aseptically removed on stainless steel table tops, and lettuce leaves are placed in clamshells.

Once packaged, they were placed into a cold room (coolbot, http://www.storeitcold.com/),

32

which is used to cool the lettuce to 41˚F. Lettuce is transported in coolers with ice to suppliers

and sanitized with oxidate between transportation to prevent cross-contamination.

Example 2: Butterhead lettuce can be grown on floating raft system with Styrofoam in a

rockwool media substance. At harvest, handlers pull out the whole lettuce head including roots,

and place them into blue Rubbermaid™ bins with water in the bottom. It is unclear if the

handlers wear gloves during harvest or packaging. Clamshells are not used for packaging. Living

lettuce heads are sold as intact with roots. Living lettuce is transported to suppliers in bins.

Example 3: Butterhead lettuce may be grown in a deepwater, floating system in a greenhouse.

However, handling has not been observed but producers market living lettuce with roots

removed through road-side farmer’s markets.

Example 4: Living lettuce can also be produced using a NFT system in an automated

greenhouse system where developing heads move on trays from one side of the greenhouse to

another according to their growth stage. These hydroponic systems have gutters but not sumps.

Gutters are soaked in oxidate for 24 hours to kill algae buildup (algae buildup can reduce the

quality of lettuce) between plantings, but the rest of the system is not cleaned. Handlers wore

gloves during harvest, but the harvest procedures were not observed. In a separate packaging

house, packing tables are cleaned with hydrogen peroxide before placing lettuce into plastic

clamshells. Packaged lettuce are placed in walk-in coolers to remove greenhouse heat for at least

24 hours before transport to retailers in coolers on ice. Coolers are sanitized after each batch of

33

lettuce is transported. This production is currently supplier to farmers’ markets and local

groceries.

Example 5: Butterhead lettuce can also be produced using simple homemade NFT hydroponic

systems in greenhouses constructed using materials from a local hardware store. It is unknown if

gutters are sanitized between plantings or what sanitation methods were using during harvest or

packaging. Living lettuce including roots were sold directly to consumers.

These variations of post-harvest practices will be valuable in developing safe agricultural

practices guidelines for hydroponic production of living lettuce. Practices designed to maintain

food safety ensures consumers access to a safe supply of leafy greens, reducing the risk of food-

borne illness outbreaks (87).

34

Table 2.4: Potential routes of transfer of Salmonella enterica in a hydroponic production.

Potential Routes of

Transfer of Salmonella species in a Hydroponic

Setting

Production

Worker's Hygiene

Plant Cultivars

Pest Infestation and Animal Feces

Water Quality for Irrigation System

Unplanned events such as system failure or floods

Harvest

Worker's Hygiene

Time and Tempereature Abuse

Cross Contamination of Harvesting Tools such as Knives

Transportation Time and Temperature Abuse

Contaminated Transport Containers

Worker's Hyigene

Retail and Home

Poor Hyigene

Sorting, Packing, Cutting, and Further Prepparation Processing can lead to Cross-

Contamination

Cross Contamination in Preparation Process such as cutting boards and knives

Time and Temperature Abuse

Water Quality for Wash and Rinse

35

Glo Germ™ as a Surrogate for Identification of Post-Harvest Cross-Contamination

Precautionary screening for bacterial contamination associated with single-food

commodities is an important practice to prevent multistate food-borne outbreaks of illness. If

protective measures are not taken, severe consequences, such as legal action against producers

or food handlers, can result when contaminated vegetables are sold that cause serious food-borne

illnesses outbreaks.

In industry, leafy greens samples are selected at random for testing. FDA’s

Bacteriological Analytical Manual (BAM) is contains standard guidelines for microbiological

analyses of food to assess safety and prevention of contamination (82). According to the FDA’s

Bacteriological Analytical Manual (BAM) Chapter 5: Salmonella, the multistep standard

guidelines that should be used in industry production to routinely screen for contamination are as

follows: 1. Aseptically weigh 25 g of leafy greens into a sterile flask, add 225 ml lactose broth

and mix thoroughly, 25 times clockwise and 25 times counterclockwise. 2. Leave the contexts in

the flask for an hour at room temperature, measure the pH and adjust to 6.8 ± 0.2 with 1N NaOH

or 1N HCl. Incubate at 35º ± 2.0º C for 24 hours. 3. Then transfer appropriate volume of contexts

to appropriate selective enrichment media and incubate plates at 35º ± 2.0º C for 24 hours. 4.

Read and record presence of colonies such as Salmonella enterica.

Screening leafy greens to detect positive Salmonella enterica or other bacteria is vital to

prevent a food-borne illness outbreak. Poor detection of contamination is a liability risk and

appropriate critical actions should be set into place to prevent the chain reaction of contamination

throughout a facility or phases of a supply chain (82). Therefore, improving the accuracy of

testing for Salmonella enterica and other human pathogens on leafy greens is important. One

potential method to improve screening of Salmonella enterica is by obtaining samples from

36

specific production regions. This information is critical for successful screening because it

allows handlers to see what the potential risk for contamination is throughout a facility.

Direct screening from specific production regions can successfully identify human

pathogens on leafy greens and potentially stop an outbreak of food-borne illness from occurring.

However, there are challenges in recovering contaminated leafy greens in terms of product age,

region of production, limit of detection, specific cultivars, environment, and the resources

available. Various examples are given below to demonstrate the difficulty in recovering

pathogens from contaminated leafy greens.

Jacobson et al. (42) investigated the three preparation methods, soak, stomach, and blend,

for the best recovery of Salmonella enterica identified from leafy green samples using the FDA’s

Bacteriological Analytical Manual (BAM) Salmonella culture method. Analysis showed

344/540 were Salmonella enterica positive when using the soaked method, 293/540 linked to

positive Salmonella by using stomaching method, and 232/540 by blending method (42). This

suggests preparation via soak method is more likely to detect Salmonella enterica from leafy

greens when compared to other common preparation methods (42). This study suggests that the

soak method may optimize the recovery of Salmonella from leafy greens.

Previous studies have investigated the potential risk associated with leaf age in the

contamination of lettuce with E. coli O157:H7 and Salmonella enterica (16, 49). It was

demonstrated that both pathogens achieved about 10-fold greater numbers on young leaves

compared to middle leaves at pre-harvest and post-harvest stages on wet leaves at 28˚C. Leaf age

and leaf region should be taken into consideration when screening for biological contamination

of lettuce (16, 49).

37

Another factor that can complicate screening is the variation of microbial load across

seasons. Prior studies have demonstrated microbial loads associated with post-harvest leafy

greens are influenced by seasons (2, 20). Statistical analysis revealed that Summer and Autumn

contain higher microbial concentrations compared to Winter and Spring seasons. These

variations in microbial loads could be impacted by drought, warm/cold temperature, moisture

content in the air, humidity, and rainy seasons (2, 20). Therefore, it is important to monitor and

consider weather conditions when screening samples. Geographic location can impact microbial

populations in addition to seasons of the years.

CONCLUSION

Further efforts are necessary to understand the preferred modes of contamination and

appropriate intervention needed to reduce the impacts/risks of food-borne outbreaks. An

understanding of how bacterial contaminations occur will enhance the microbiological safety of

leafy greens in a hydroponic production setting. Post-harvest contamination during handling is a

potential source and understanding the risks of handling leafy greens is critical to maintaining

safe food practices. Producers should identify the risks associated and implement an action plan

to reduce microbial contaminations at its critical points. The identification of risks and

development of a plan should be documented and executed to ensure preventive methods that are

most effective since washing cannot completely eliminate bacterial contamination. The

importance of contamination prevention must be emphasized at all stages of production

including harvesting, processing, storage and preparation of leafy greens; this contributes to the

safety of leafy greens and economic value of the food industry. It also ensures consumers access

to a safe food supply of fresh leafy greens and protects the well being of the consumers resulting

in an overall lower risk of food-borne illness outbreaks. Assessing the continuation of reports of

38

food-borne disease outbreaks is critical towards the success of current and future prevention

strategies. These insights will be valuable in developing safe agricultural practices for pre- and

post-harvest of leafy greens in a hydroponic production. Hydroponics can function as an

alternative agricultural practice that can be sustainable for the production of leafy greens.

39

REFERENCES

1. Ahmer, B. M. 2004. Cell-to-cell signalling in Escherichia coli and Salmonella enterica.

Molecular Microbiology. 52:933-45.

2. Ailes, E. C., J. S. Leon, L.A. Jaykus, L. M. Johnston, H. A. Clayton, S. Blanding, D. G.

Kleinbaum, L. C. Backer, and C. L. Moe. 2008. Microbial concentrations on fresh produce are

affected by post-harvest processing, importation, and season. Journal of Food Protection.

71:2389-2397.

3. Anonymous. 2005. Ohio specialty crop food safety initiative greenhouse food safety

manual. Pg number?

4. Appel, L. J., T. J. Moore, E. Obarzanek, W. M. Vollmer, L. P. Svetkey, F. M. Sacks, G.

A. Bray, T. M. Vogt, J. A. Cutler, M. M. Windhauser, P.H. Lin, N. Karanja, D. Simons-Morton,

M. McCullough, J. Swain, P. Steele, M. A. Evans, E. R. Miller, and D. W. Harsha. 1997. A

clinical trial of the effects of dietary patterns on blood pressure. New England Journal of

Medicine. 336:1117-1124.

5. Barak, J. D., and B. K. Schroeder. 2012. Interrelationships of food safety and plant

pathology: The life cycle of human pathogens on plants. Annual Review of Phytopathology.

50:12.1–12.26

6. Barak, J. D., L. Gorski, P. Naraghi-Arani, and A. O. Charkowski. 2005. Salmonella

enterica virulence genes are required for bacterial attachment to plant tissue. Applied and

Environmental Microbiology. 71:5685-5691.

7. Barak, J. D., C. E. Jahn, D. L. Gibson, and A. O. Charkowski. 2007. The role of cellulose

and o-antigen capsule in the colonization of plants by Salmonella enterica. Molecular Plant-

Microbe Interactions. 20:1083-1091.

8. Bartz, J., and J. Brecht. 2003. Sales of vegetables for fresh market: The requirement for

hazard analysis and critical control points (HACCP) and sanitation. Post-harvest physiology and

pathology of vegetables. Marcel dekker, Inc, New York, USA. P.563-580.

9. Batz, M., S. Hoffmann and J.G. Morris. 2011. Ranking the risks the 10 pathogen-food

combination with the greatest burden on public health. Emerging Pathogens Institute University

of Florida. Available at: https://folio.iupui.edu/bitstream/handle/10244/1022/72267report.pdf

Accessed September 2012.

10. Bertin, C., X. Yang, and L. Weston. 2003. The role of root exudates and allelochemicals

in the rhizosphere. Plant and Soil. 256:67-83.

11. Beuchat, L. R. 2002. Ecological factors influencing survival and growth of human

pathogens on raw fruits and vegetables. Microbes and Infection. 4:413-423.

40

12. Beuchat, L. R. 1996. Pathogenic microorganisms associated with fresh produce. Journal

of Food Protection. 59:204-216.

13. Beuchat, L. R. World health organization.1998. Surface decontamination of fruits and

vegetables eaten raw: A review.Available at:

http://www.who.int/foodsafety/publications/fs_management/surfac_decon/en/. Accessed August

2012.

14. Blaser, M. J., and L. S. Newman. 1982. A review of human Salmonellosis: I. Infective

dose. Reviews of Infectious Diseases. 4:1096-1106.

15. Bradley, P., and C. Marulanda. 2001. Simplified hydoponics to reduce global hunger.

ISHA Acta Horticulturae. 554:289-296.

16. Brandl, M. T., and R. Amundson. 2008. Leaf age as a risk factor in contamination of

lettuce with Escherichia coli O157:H7 and Salmonella enterica. Applied and Environmental

Microbiology. 74:2298-2306.

17. Brecht, J. K. 1993. Physiology of lightly processed fruits and vegetables. HortScience.

28:472.

18. Buck, J. W., Walcott, R.R., Beuchat, L.R. 2003. Recent trends in microbiological safety

of fruits and vegetables. Plant Health Progress.10:0121-0131.

19. Burnett, S. L., and L. R. Beuchat. 2000. Human pathogens associated with raw produce

and unpasteurized juices, and difficulties in decontamination. Journal of Industrial Microbiology

& Biotechnology. 25:281.

20. Caponigro, V., M. Ventura, I. Chiancone, L. Amato, E. Parente, and F. Piro. 2010.

Variation of microbial load and visual quality of ready-to-eat salads by vegetable type, season,

processor and retailer. Food Microbiology. 27:1071-1077.

21. Centers of Disease Control and Prevention. 2011. CDC estimates of food-borne illness in

the United States. Available at:

http://www.cdc.gov/foodborneburden/PDFs/FACTSHEET_A_FINDINGS_updated4-13.pdf.

Accessed July 2012.

22. Centers of Disease Control and Prevention. 2006. FDA statement on food-borne E. coli

O157:H7 outbreak in spinach Available at:

http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/2006/ucm108761.htm.

Accessed July 2012.

23. Centers of Disease Control and Prevention. 2011. Investigation announcement: multistate

outbreak of E. coli O157:H7 infections linked to romaine lettuce: December 7, 2011. Available

at: http://www.cdc.gov/ecoli/2011/ecoliO157/romainelettuce/120711/index.html. Accessed July

2012.

41

24. Centers for Disease Control and Prevention. Multistate food-borne outbreaks

investigations. Available at: http://www.cdc.gov/salmonella/braenderup-08-12/index.html,

http://www.cdc.gov/ecoli/2011/ecoliO157/romainelettuce/120711/index.html,

http://www.cdc.gov/salmonella/newport/index.html,

http://www.cdc.gov/salmonella/litchfield/,

http://www.cdc.gov/ncidod/dbmd/diseaseinfo/salmonellosis_2006/110306_outbreak_notice.htm,

http://www.cdc.gov/foodborne/ecolispinach/100606.htm. Accessed October 21, 2012.

25. Centers of Disease Control and Prevention. 1998. Outbreak of Campylobacter Enteritis

associated with cross-contamination of food-oklahoma, 1996. Available at:

http://www.cdc.gov/mmwr/preview/mmwrhtml/00051427.htm. Accessed July 2012.

26. Centers of Disease Control and Prevention. 2011. Trends in food-borne illness, 1996-

2010. Available at:

http://www.cdc.gov/foodborneburden/PDFs/FACTSHEET_B_TRENDS.PDF. Accessed July

2012.

27. Cooley, M., D. Carychao, L. Crawford-Miksza, M. T. Jay, C. Myers, C. Rose, C. Keys, J.

Farrar, and R. E. Mandrell. 2007. Incidence and tracking of Escherichia coli O157:H7 in a major

produce production region in california. PLoS ONE. 2:e1159.

28. Davis, H., J. P. Taylor, J. N. Perdue, G. N. Stelma, R. Rowntree, and K. D. Greene. 1988.

A Shigellosis outbreak traced to commerically distributed shredded lettuce. American Journal of

Epidemiology. 128:1312-1321.

29. Delaquis, P., S. Bach, and L.D. Dinu. 2007. Behavior of Escherichia coli O157:H7 in

leafy vegetables. Journal of Food Protection. 70:1966-1974.

30. Dickenson County Discussion Board. Hydroponics. Available at:

http://appalachianforums.com/forums/Dickenson_County,_Virginia.pl/md/read/id/260519.

Accessed April 25, 2013.

31. Doyle, M. P., and M. C. Erickson. 2008. Summer meeting 2007 the problems with fresh

produce: an overview. Journal of Applied Microbiology. 105:317-330.

32. Fallovo, C., Y. Rouphael, E. Rea, A. Battistelli, and G. Colla. 2009. Nutrient solution

concentration and growing season affect yield and quality of Lactuca sativa L. var. acephala in

floating raft culture. Journal of the Science of Food and Agriculture. 89:1682-1689.

33. Food and Agriculture Organization of the United Nations. World Health Organization.

2008. Microbiological risk assessment series. Microbiological hazards in fresh fruits and

vegetables: meeting report. Available at:

http://whqlibdoc.who.int/publications/2008/9789241563789_eng.pdf. Accessed September 2012.

42

34. Gallegos-Robles, M. A., A. Morales-Loredo, G. Alvarez-Ojeda, A. Vega-P, Y. Chew-M,

S. Velarde, and P. Fratamico. 2008. Identification of Salmonella serotypes isolated from

cantaloupe and chile pepper production systems in Mexico by PCR Restriction fragment length

polymorphism. Journal of Food Protection. 71:2217-2222.

35. George, R. 2009. Vegetable seed production. MPG Books Group, Bodmin, UK.

36. Grewal, H. S., B. Maheshwari, and S. E. Parks. 2011. Water and nutrient use efficiency

of a low-cost hydroponic greenhouse for a cucumber crop: An Australian case study.

Agricultural Water Management. 98:841-846.

37. Hammack, T. 2012. Bad bug book-food-borne pathogenic microorganisms and natural

toxins-Salmonella species. Available at:

http://www.fda.gov/downloads/Food/FoodSafety/FoodborneIllness/FoodborneIllnessFoodborneP

athogensNaturalToxins/BadBugBook/UCM297627.pdf. Accessed August 2012.

38. Herman, K. M., T.L. Ayers, and M. Lynch. 2008. Foodborne disease outbreaks

associated with leafy greens 1973-2006. International Conference on Emerging Infectious

Disease.

39. Hong, C. X., and G. W. Moorman. 2005. Plant Pathogens in Irrigation Water: Challenges

and Opportunities. Critical Reviews in Plant Sciences. 24:189-208.

40. Hydroponic Crop Farming in the US: Market Research Report. Available at:

http://www.ibisworld.com/industry/hydroponic-crop-farming.html. Accessed April 25th, 2013.

41. Islam, M., J. Morgan, M. Doyle, S. Phatak, P. Millner, and X. Jiang. 2004. Persistence of

Salmonella enterica serovar Typhimurium on lettuce and parsley and in soils on which they were

grown in fields treated with contaminated manure composts or irrigation water. Foodborne

Pathogens and Diseases. 1:27-35.

42. Jacobsen, C. S., and T. B. Bech. 2012. Soil survival of Salmonella and transfer to

freshwater and fresh produce. Food Research International. 45:557-566.

43. Jay, M.T., M. Cooley, D. Carychao, G.Wiscomb, R.A. Sweitzer, L. Crawford-Miksza, et

al. 2007. Escherichia coli O157:H7 in feral swine near spinach fields and cattle, central

California coast. Available at: http://wwwnc.cdc.gov/eid/article/13/12/07-0763.htm. Accessed

September 2012.

44. Jacobson, A. P., V. S. Gill, K. A. Irvin, H. Wang, and T. S. Hammack. 2012. Evaluation

of methods to prepare samples of leafy green vegetables for preenrichment with the

bacteriological analytical manual Salmonella culture method. Journal of Food Protection.

75:400-404.

43

45. Kisluk, G., and S. Yaron. 2012. Presence and persistence of Salmonella enterica serotype

Typhimurium in the phyllosphere and rhizosphere of spray-irrigated parsley. Applied and

Environmental Microbiology. 78:4030-4036.

46. Klerks, M. M., E. Franz, M. van Gent-Pelzer, C. Zijlstra, and A. H. C. van Bruggen.

2007. Differential interaction of Salmonella enterica serovars with lettuce cultivars and plant-

microbe factors influencing the colonization efficiency. Internaitonal Society Microbial Ecology

Journal. 1:620-631.

47. Klerks, M. M., M. van Gent-Pelzer, E. Franz, C. Zijlstra, and A. H. C. van Bruggen.

2007. Physiological and molecular responses of Lactuca sativa to colonization by Salmonella

enterica serovar Dublin. Applied and Environmental Microbiology. 73:4905-4914.

48. Koseki, S., Y. Mizuno, and K. Yamamoto. 2011. Comparison of two possible routes of

pathogen contamination of spinach leaves in a hydroponic cultivation system. Journal of Food

Protection. 74:1536-1542.

49. Kroupitski, Y., R. Pinto, E. Belausov, and S. Sela. 2011. Distribution of Salmonella

Typhimurium in romaine lettuce leaves. Food Microbiology. 28:990-997.

50. Kroupitski, Y., R. Pinto, M. T. Brandl, E. Belausov, and S. Sela. 2009. Interactions of

Salmonella enterica with lettuce leaves. Journal of Applied Microbiology. 106:1876-1885.

51. Lapidotand, A., and S. Yaron. 2009. Transfer of Salmonella enterica serovar

Typhimurium from contaminated irrigation water to parsley is dependent on curli and cellulose,

the biofilm matrix components. Journal of Food Protection. 72:618-623.

52. Leveau, J. 2009. Microbiology: Life on leaves. Nature. 461:741-742.

53. Lewis, D. J., E. R. Atwill, M. S. Lennox, L. Hou, B. Karle, and K. W. Tate. 2005.

Linking on-farm dairy management practices to storm-flow fecal coliform loading for California

coastal watersheds. Environmental Monitoring and Assessment. 107:407-425.

54. Lynch M.F., Tauxe R. V., and C. W. Hedberg. 2009. The growing burden of food-borne

outbreaks due to contaminated fresh produce: risks and opportunities. Epidemiology and

Infection. 137:307-315.

55. Molitor, H. 1990. The European perspective with emphasis on subirrigation and

recirculation of water and nutrients. Acta Horticulturae. (ISHS). 272:165-174.

56. Moore, C. M., B. W. Sheldon, and L. A. Jaykus. 2003. Transfer of Salmonella and

Campylobacter from stainless steel to romaine lettuce. Journal of Food Protection. 66:2231-

2236.

44

57. Morgan, L. 1999. Hydroponic lettuce production a comprehensive, practical and

scientific guide to commerical hydroponic lettuce production. Casper Publications Pty Ltd

Narrabeen, New Zealand.

58. Mou, B. 2008. Vegetables I asteraceae, brassicaceae, chenopodicaceae, and

cucurbitaceae. Springer Science Business Media, LLC, New York, NY, USA.

59. Natvig, E. E., S. C. Ingham, B. H. Ingham, L. R. Cooperband, and T. R. Roper. 2002.

Salmonella enterica serovar Typhimurium and Escherichia coli contamination of root and leaf

vegetables grown in soils with incorporated bovine manure. Applied and Environmental

Microbiology. 68:2737-2744.

60. Nicola, S., J. Hoeberechts, and E. Fontana. 2005. Comparsion between traditional and

soilless culture systems to produce rocket (Eruca sativa) with low nitrate concent. ISHA Acta

Horticulturae. 697:549-555.

61. Nygård, K., J. Lassen, L. Vold, Y. Andersson, I. Fisher, S. Löfdahl, J. Threlfall, I. Luzzi,

T. Peters, M. Hampton, M. Torpdahl, G. Kapperud, and P. Aavitsland. 2008. Outbreak of

Salmonella Thompson infections linked to imported Rucola Lettuce. Foodborne Pathogens and

Diseases. 5:165-173.

62. Oliveira, M., J. Usall, C. Solsona, I. Alegre, I. Viñas, and M. Abadias. 2010. Effects of

packaging type and storage temperature on the growth of food-borne pathogens on shredded

romaine lettuce. Food Microbiology. 27:375-380.

63. Patel, J., and M. Sharma. 2010. Differences in attachment of Salmonella enterica

serovars to cabbage and lettuce leaves. International Journal of Food Microbiology. 139:41-47.

64. Personal communication with hydroponic lettuce grower. 2011. Blacksburg, Virginia.

65. Runia, W.T. 1994. Elimination of root-infecting pathogens in recirculation water from

closed cultivation systems by ultra-violet radiation. Acta Horticulturae. (ISHS). 361:361-371.

66. Sapers, G. 2001. Efficacy of washing and sanitizing methods for disinfection of fresh

fruit and vegetable products. Food Technology and Biotechnology. 39:305-311.

67. Scallan E, H. R., Angulo FJ, Tauxe RV, Widdowson M.A, Roy SL. 2011. Food-borne

illness acquired in the United States-major pathogens. Emerging Infectious Diseases.17.

68. Scharff, R. L. 2010. Health-related costs from food-borne illness in the united states. The

produce safety project at georgetown university. www.producesafetyproject.org. Accessed July

2012.

69. Selma, M., M. Luna, A. Martínez-Sánchez, J. Tudela, D. Beltrán, C. Baixauli, and M.

Gil. 2012. Sensory quality, bioactive constituents and microbiological quality of green and red

45

fresh-cut lettuces (Lactuca sativa l.) are influenced by soil and soilless agricultural production

systems. Postharvest Biology and Technology. 63:16-24.

70. Shirron, N., and S. Yaron. 2011. Active suppression of early immune response in tobacco

by the human pathogen Salmonella typhimurium. PLoS ONE. 6:18855.

71. Solomon, E. B., S. Yaron, and K. R. Matthews. 2002. Transmission of Escherichia coli

O157:H7 from contaminated manure and irrigation water to lettuce plant tissue and its

subsequent internalization. Applied and Environmental Microbiology. 68:397-400.

72. Stafford, R. J., McCall, B.J., Neill, A.S., Leon, D.S., Dorricott, G.J., Towner, C.D. and

Micalizz, G.R. 2002. A statewide outbreak of Salmonella Bovismorbificans phage type 32

infection in Queenland. Communicable Diseases Intelligence Quartely Report. 26:568-573.

73. Sumathi, S., R. F. Cindy, C. Linda, and V. T. Robert. 2004. Fresh produce: A growing

cause of outbreaks of food-borne illness in the United States, 1973 through 1997. Journal of

Food Protection. 67:2342-2353.

74. Suslow, T. 2010. Produce safety project issue brief: standards for irrigation and foliar

contact water. Produce Safety Project, Georgetown University, Washington, DC.

75. Sutton, J.C., Yu, H., Grodzinski, B., Johnstone, M. 2000. Relationships of ultraviolet

radition dose and inactivation of pathogen propagules in water and hydroponic nutrient solutions.

Canadian Journal of Plant Pathology. 22:300-309.

76. Tyrrel, S. F., J. W. Knox, and E. K. Weatherhead. 2006. Microbiological water quality

requirements for salad irrigation in the United Kingdom. Journal of Food Protection. 69:2029-

2035.

77. U.S. Department of Agruiculture. Economic Research Service. 1989-2009. All lettuce

U.S. imports from selected countries. Available at:

http://usda.mannlib.cornell.edu/MannUsda/viewDocumentInfo.do?documentID=1576. Accessed

July 2012.

78. U.S. Food and Drug Administration. 2009. Archive for recalls, market withdrawals &

safety alerts: foods. Available at: http://www.fda.gov/Safety/Recalls/ArchiveRecalls/default.htm.

Accessed September 2012.

79. U.S Food and Drug Administration. 2009. Archive for recalls, market withdrawals &

safety alerts: foods. Recall: AZ hydroponic farming recalls 4oz alfalfa sprout cup because of

possible health risk. Available at: http://www.fda.gov/safety/recalls/ucm148389.htm. Accessed

November 2012.

80. U.S Food and Drug Administration. 2012. Archive for recalls, market withdrawals &

safety alerts: foods. Recall: Dole fresh vegetables announces precautionary recall of limited

46

number of salads. Available at: http://www.fda.gov/Safety/Recalls/ucm300414.htm. Accessed

November 2012.

81. U.S. Food and Drug Administration. 2006. Commodity specific food safety guidelines

for the lettuce and leafy greens supply chain. Available at:

http://www.fda.gov/downloads/Food/FoodSafety/ProductSpecificInformation/FruitsVegetablesJ

uices/GuidanceComplianceRegulatoryInformation/UCM169008.pdf. Accessed September 2012.

82. U.S. Food and Drug Administration. 2011. FDA’s bacteriological analytical manual

(BAM) chapter 5: Salmonella. Available at:

http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManua

lBAM/ucm070149.htm. Accessed September 2012.

83. U.S. Food and Drug Administration. 2008. Guidance for industry: guide to minimize

microbial food safety hazards of fresh-cut fruits and vegetables. Available at:

http://www.fda.gov/food/guidancecomplianceregulatoryinformation/guidancedocuments/produce

andplanproducts/ucm064458.htm. Accessed September 2012.

84. U.S. Environmental Protection Agency. 2004. The national water quality inventory:

Report to congress for the 2004 reporting cycle-a profile. Available at:

http://water.epa.gov/lawsregs/guidance/cwa/305b/upload/2009_01_22_305b_2004report_factshe

et2004305b.pdf. Accessed October 2012.

85. U.S. Department of Agruiculture. Economic Research Service. 1960-2010. U.S. lettuce

per capita. Available at:

http://usda.mannlib.cornell.edu/MannUsda/viewDocumentInfo.do?documentID=1576. Accessed

July 2012.

86. U.S. Department of Agruiculture. Economic Research Service. 2009. U.S. lettuce

production value. Available at:

http://usda.mannlib.cornell.edu/MannUsda/viewDocumentInfo.do?documentID=1212. Accessed

July 2012.

87. Vallatton, A. 2012. Personal communication with Amber Vallatton, Virginia cooperative

extension agent, Rockbridge County on October 04, 2012.

88. Wachtel, M. R., and A. O. Charkowski. 2002. Cross-contamination of lettuce with

Escherichia coli O157:H7. Journal of Food Protection. 65:465-470.

89. Weissinger, W. R., W. Chantarapanont, and L. R. Beuchat. 2000. Survival and growth of

Salmonella Baildon in shredded lettuce and diced tomatoes, and effectiveness of chlorinated

water as a sanitizer. International Journal of Food Microbiology. 62:123-131.

90. Wells, J. M., and J. E. Butterfield. 1997. Salmonella contamination associated with

bacterial soft-rot of fresh fruits and vegetables in the marketplace. Plant Disease. 81:867-872.

47

91. Zhang, G., L. Ma, L. R. Beuchat, M. C. Erickson, V. H. Phelan, and M. P. Doyle. 2009.

Evaluation of treatments for elimination of food-borne pathogens on the surface of leaves and

roots of lettuce (Lactuca sativa l.). Journal of Food Protection. 72:228-34.

48

CHAPTER 3: GLO GERM™ AS A SURROGATE FOR IDENTIFICATION OF POST-

HARVEST CROSS-CONTAMINATION ON LIVING LETTUCE

ABSTRACT

Fresh produce has been associated with outbreaks of Salmonellosis with increasing

frequency. Pre-harvest risk factors have been identified for contamination of field-harvested

lettuce; however, the transmission of human pathogens to hydroponic lettuce is not well

understood. The purpose of our study was to investigate the use of Glo Germ™, a fluorescent

microsphere solution, as a method to visualize cross-contamination from worker hands to the

edible tissue of living lettuce. Glo Germ™ solution was combined with Salmonella enterica

serotype Enteritidis and applied to handlers’ hands to identify areas of contact between the

solution and the lettuce, providing information about potential for cross-contamination.

The effectiveness of current sampling strategies for detection of Salmonella were

evaluated by comparing randomly selected leaves under blacklight for Glo Germ™ presence.

Living lettuce heads were sequentially processed using typical handling practices, leaves were

randomly selected from different sections of the head, homogenized in peptone water, and

Salmonella were enumerated through serial dilution and plating onto XLT-4 agar. Edible leaves

with multiple glowing areas activated under blacklight were selected and processed using the

method described previously. On average 4.6±0.06 log CFU/g of Salmonella were removed from

leaves selected under blacklight compared to 4.1±0.09 log CFU/g from randomly chosen leaves

(P=0.05). Although our study suggests that the current FDA methodology for selection of leaves

from lettuce heads is adequate, we believe that Glo Germ™ has the potential to be used as a

49

teaching aid to promote safe handling awareness of leafy greens and minimize cross-

contamination risks.

50

INTRODUCTION

Food-borne illness outbreaks associated with consumption of contaminated fresh

produce are increasing in frequency. Produce-associated outbreaks reported by Centers for

Disease Control and Prevention (CDC) increased from 2 outbreaks per year in the 1970s to 16

per year in the 1990s (23). Food-borne outbreaks associated with leafy greens reported from

1996 to 2005 have increased by 38.6% (11). Between 1973 and 2006, a total of 502 food-borne

outbreaks associated with leafy greens were reported, and of those, 35 were attributed

Salmonella (11). Between 1998 and 2008, leafy greens were associated with 186,140 illnesses

from bacterial agents, 2,367 hospitalizations, and 26 deaths (20). Human pathogens can be

introduced at any point throughout the farm to fork continuum, and the scale of contamination

can be amplified through post-harvest handling activities like washing, handling and packing (4,

7, 17, 21). Improved risk-based interventions applied to produce harvesting, transportation, and

handling are important to minimize risk of cross-contamination in controlled greenhouse

settings.

Consumption of “living lettuce”, lettuce heads with intact roots packed in plastic

clamshells, is increasing in the United States. Typically, living lettuce is produced

hydroponically within controlled greenhouses which reduce potential exposure to wildlife and

run-off associated contamination. However, other potential sources of contamination do exist,

such as cross-contamination from infected handler to the edible leaves. The purpose of this

experiment was to investigate Glo Germ™, a fluorescent microsphere solution, as a method to

visualize cross-contamination from worker hands to the edible leaves of living lettuce. Glo

Germ™ combined with Salmonella enterica serotype Enteritidis was applied to the handlers’

hands to identify sections of the lettuce head that are more susceptible to handler-associated

51

contamination. The effectiveness of lettuce sampling strategies were examined by looking at

recoverability of Salmonella Enteritidis from randomly selected leaves versus leaves chosen

using a blacklight as a tool to identify potential cross-contamination. This evaluates the

effectiveness of current sampling methodology in industries.

In industry, FDA’s (U S Food and Drug Administration) Bacteriological Analytical

Manual (BAM) Chapter 5: Salmonella, is the current methodology of detecting Salmonella spp.

on leafy greens (25). Lettuce leaves are randomly selected from the head during routine

precautionary screening for human pathogens (25). Current methodology of random selection

may be inadequate due to uneven distribution on the head or ineffective screening. Leafy greens

are known to carry Salmonella, therefore the FDA’s Bacteriological Analytical Manual (BAM)

Chapter 5: Salmonella method’s effectiveness for leafy greens need to be reexamined.

Salmonella isolation from various food commodities has been done using different culture

media, incubation temperatures, and sample preparation (3, 8-10, 12). We hypothesize that

sampling efficiency might be improved by targeting specific portions of lettuce (outer vs. inner

towards the head or bottom vs. top of leaf), which may be more likely to come into contact with

hands of handlers. This informative screening has the potential to aid handlers in visualizing

potential contamination risks and promote awareness with facilities.

MATERIALS AND METHODS

Bacterial strain and culture conditions.

Salmonella enterica Enteritidis ptvs177 was obtained from Trevor Suslow of University

of California, Davis and passed through increasing concentrations of rifampicin to generate

resistance to 100 µg/ml rifampicin (Fisher Scientific, Fair Lawn, NJ). Bacteria were stored at -

80˚C in tryptic soy broth (TSB; Difco) supplemented with 100 µg/ml rifampicin (TSB-Rif) and

52

30% glycerol (Fisher Scientific). Bacterial cultures were prepared by sub-culturing from a

frozen stock in TSB-Rif 100 µg/ml and incubated at 37˚C for 24 h. Colony morphology and

selective indicators were confirmed by plating onto xylose-lysine-tergitol-4-agar (XLT-4; Difco)

containing 100 µg/ml rifampicin (XLT-4- Rif) and incubated statically at 37˚C for 24 h.

Salmonella colonies were identified by their red coloring with black centers. Enumeration of

Salmonella CFU/g was performed using a 10-fold serial dilution in 0.1% sterile peptone water

solution (BPW; Sigma-Aldrich) and were spread plated onto duplicate XLT-4 plates. Plates were

incubated at 37˚C for 48 h.

Inoculum preparation and Glo Germ™ solution preparation.

A sterile disposable plastic loop was used to transfer cells from frozen stocks of

Salmonella enterica Enteritidis to 100 ml TSB-Rif broth. Salmonella cultures were incubated at

37˚C for 24 h. Salmonella cells in 0.50 ml of TSB-Rif were washed with 0.1% sterile BPW to

remove residual nutrients. After final centrifugation at 3,000 x g for 15 min, the cell pellet was

re-suspended in 0.5 ml BPW, and Glo Germ™ solution was added for a total final volume of

12.5 ml. A ratio of 1g Glo Germ™ to 11 ml of 0.1% BPW, the culture was re-suspended in Glo

Germ™ solution to create a final 6.5-7 log CFU/g Salmonella.

Lettuce preparation and sampling methods.

Butterhead living lettuce heads were purchased from a local retail store in Blacksburg,

VA and kept at 4˚C for a maximum of 48 h prior to processing. Spic and Span™ latex rubber

gloves (long cuff latex) purchased from a local retail store were used to mimic the physical

texture of gloves commonly used to handle leafy greens in the production facilities. To

determine the potential transference of Salmonella from one living lettuce head to another in

sequential handling, 1.0 ml of Glo Germ™-Salmonella solution was applied via droplets onto

53

each hand. Once the Glo Germ™-Salmonella solution was applied onto the hands, then typical

living lettuce handling tasks were performed: lift a head of lettuce by the base, squeeze excess

water from roots, wrap the roots in a knot and transfer lettuce head to a plastic clamshell.

Potential transfer of Salmonella from a single contamination event was assessed by

performing sequential handling on three heads of lettuce with hands inoculated at the beginning

of the sequence. Food coloring was used to provide a visual indicator of where gloves tend to

make contact with surfaces during post-harvest handling. Results from this experiment informed

the application of Glo Germ™-Salmonella solution to the gloves during further experimental

procedures.

Living lettuce heads (n=3) were sequentially handled, and 25 g of randomly selected

leaves were weighted in a stomacher bag and the contents were homogenized for 2 min in 225

ml containing 0.1% BPW and 0.1% Tween80 (Fisher Scientific) solution in a lab blender (Bag

Mixer, Interscience, Weymouth, MA). Enumeration of Salmonella CFU/g were determined by

performing a 10-fold serial dilution in 0.1% BPW and spread plating onto duplicate XLT-4

plates. Plates were incubated at 37˚C for 48 h. The post-harvest handling study was conducted in

triplicate, using 3 heads of living lettuce for each trial. Additionally, living lettuce heads (n=3)

were sequentially handled and edible leaves with multiple glowing specs activated under

blacklight were aseptically removed. Leaves were selected and processed using previously

described method. To validate the presence of bacteria that were below limit of detection in plate

counts, 1.0 ml of homogenized contents (leaves) from stomacher bag were added to 9 ml of TSB

supplemented with rifampicin and incubated at 37˚C for 24 h. After 24 h the culture was streaked

for isolation on XLT-4-Rif and incubated at 37˚C for 24 h. Presence or absence of colonies on

each selective plate were recorded as positive or negative, respectively. Non-inoculated controls

54

(n=3) were processed as above to account for contamination or growth of non-Salmonella

bacteria on the selective media.

Quantification of Glo Germ™ transmission to edible leaves.

Presence/absence of the non-inoculated Glo Germ™ solution on the older and outer

leaves were visualized using the Gel Doc UV light box and the average intensity (Average

Quantity) within a 30x30 mm area calculated using the Quantity One 1-D Analysis Software

version 4.6.5 (Bio-Rad Laboratories, Inc. ™). The average intensity value of the leaf without

Glo Germ™ was subtracted from the average intensity value of the leaf with Glo Germ™ to

determine the fluorescence only by Glo Germ™. The Glo Germ™ was put into solution and then

the smallest detectable amount was evaluated. The duration of fluorescence was also determined

over a 24 h period. To improve the distribution of Glo Germ™ within the solution, 1.0% of

weight per volume lecithin (0.12 g of lecithin) was added to 1:12.5 ml Glo Germ™ solution and

heated to 30˚C before application.

Growth of Salmonella ptvs177 in presence of Glo Germ™ solution.

The Bioscreen C™ (Growth Curve, Inc.) instrument was used to determine the growth

rate of Salmonella in a TSB/Glo Germ™ solution. Wells containing 178.6 µl TSB-Rif, 20 µl,

Glo Germ™ solution were inoculated with 1.4 µl of Salmonella. Shaking occurred 15 sec before

each absorbance measurements (OD580 nm) reading recorded every 15 min for 48 h at 37˚C.

Concentrations of Salmonella containing 20 µl, BPW, 1.4 µl Salmonella and TSB supplemented

with rifampicin 178.6 µl, total volume of 200 µl, were pipette into 10 wells to serve as a positive

control of growth. Negative controls consisted of growth media to which bacteria were not

added. Microsoft Office Excel 2007 software was used to plot absorbance vs. time (hours) to

identify and calculate the lag phase, starting phase, exponential phase, slowing down phase,

55

stationary phase, and the growth rate constant. Growth curves were run in duplicate using 40

wells.

Statistical analysis.

Bacterial counts (CFU/g) were log transformed to approximate normal distribution. The

post-harvest sequential handling and selective method study was conducted three times using six

living lettuce heads per trial. Statistical analyses were performed using the using statistical

software JMP® Pro 10.0 (SAS; Institute Inc., Cary, NC). Fisher’s exact two-tailed F test was

performed to test for differences in the bacterial counts and growth rate. The sequential handling

was statistically investigated by analysis of variance and Student’s t test. P-values (P< 0.05) with

=0.5 were considered significant.

RESULTS

Testing efficacy of sampling plans for detection of Salmonella on living lettuce.

The average log CFU/g of Salmonella recovered from lettuce leaves chosen using two

different sampling strategies were compared. On average 4.6±0.1 log CFU/g of Salmonella

detected under blacklight and 4.1±0.1 log CFU/g recovered from randomly selected leaves. We

observed a small but statistically significant increase in log CFU/g of Salmonella recovered

when blacklight was used to select leaves (P=0.05) (Figure 3.4). After each sequential handling

event, the log CFU/g of Salmonella was reduced. Statistically significant was observed between

lettuce head 1 and lettuce head 3 (P=0.0486) selected under blacklight and randomized leaves

(P=0.0002). The average recovery of Salmonella from leaves selected under blacklight on

lettuce head 1 was 4.8±0.1 log CFU/g, lettuce head 2 was 4.6±0.0 log CFU/g, and lettuce head 3

was 4.2±0.1 log CFU/g. The average recovery of Salmonella from leaves randomly selected on

lettuce head 1 was 4.7±0.1 log CFU/g, lettuce head 2 was 4.1±0.1 log CFU/g, and lettuce head 3

56

was 3.4±0.1 log CFU/g (Figure 3.5). Growth rates (μ=0.79) of Salmonella in the presence of Glo

Germ™ solution at 37˚C were not significantly different from the control. However, the overall

final yield (maximum OD) of Glo Germ™ without the presence of Salmonella (OD 580nm= 1.1)

was greater than Glo Germ™ containing Salmonella (OD 580nm= 0.8). This suggests Glo

Germ™ solution may influence the overall amount of Salmonella at 37˚C. Non-inoculated

controls (n=3) yield no counts.

Use of Glo Germ™-Salmonella solution to visualize transfer from gloves to living lettuce.

Prior to applying Glo Germ™-Salmonella solution, food coloring was used as a visual

guide to determine where the gloves came into contact during post-harvest handling of living

lettuce. These results led to a better application of Glo Germ™-Salmonella solution on the

gloves to assure contact was made with the solution. Food coloring was visible on the roots,

base of the lettuce head and tips of leaves (Figure 3.1). Blacklight revealed distribution of small

droplets of Glo Germ™- Salmonella solution on the roots, and over the outer leaves and across

the top of the lettuce heads, including areas that were not directly touched by the handler (Figure

3.2). The use of the average intensity function of the Quantity One program was insufficient to

quantify the amount of transference from workers hands to leaves. The readings were

inconsistent and frequently the average intensity of the background, non-inoculated leaves prior

to application of Glo Germ™ solution was greater than that measured for the leaves that were

inoculated with the Glo-germ solution, in addition the inoculated leaves appeared brighter to the

researchers (Figure 3.3).

DISCUSSION

Glo Germ™ is a useful tool for monitoring Salmonella transmission routes and assessing

hygiene practices and identifying control points during post-harvest handling. In this study,

57

simultaneous application of Glo Germ™ and Salmonella was used to visualize the sections of

lettuce that are subject to contamination via handler’s hands. We observed only a small, 0.5 log

CFU/g of Salmonella on leaves that were selected with aid of blacklight to visualize potential

contamination compared to leaves that were chosen at random. This supports our hypothesis that

the current FDA protocol of randomized leaf sampling in multiple locations in production is

adequate. The initial concentration of Salmonella applied to the worker hands was 6.5 log

CFU/ml, an amount that is likely to be higher than real life contamination events. However, this

technique demonstrated that while large numbers 4.0-4.8 log CFU/g were transferred to the

lettuce, enough remained on worker hands for transmission to multiple heads of lettuce. While

this experiment examined sequential transfer to only 3 heads, the large numbers on head three

suggest transfer occurs to even more heads. Taormina et al. (24) have demonstrated that E. coli

O157:H7 can be transferred from knives to lettuce during field coring and a single contamination

event can spread the pathogen to 10 heads. Given that the infectious dose of Salmonella may be

as low as 100 cells of Salmonella dependent on age, immunization status, and serotypes,

sequential transfer after only one contamination event may pose significant risk to consumers

(1). Transfer of human pathogens from human hands to surfaces and to produce has been

demonstrated for Salmonella, E.coli and Campylobacter (5, 16, 27). Wachtel et al. (27)

examined the cross-contamination risk of ‘Iceberg’ lettuce associated with contaminated hands

and cutting boards. Results revealed 46% of lettuce leaves, including the 25th

exposed leaf were

contaminated when the leaves were pressed onto a cutting board inoculated with 1.25 X 102

CFU

of Escherichia coli O157:H7 (27). Chen et al. (5) reported cross-contamination risk was

significant when raw chicken was inoculated with 7 log CFU/g of Enterobacter aerogenes and

demonstrated that 2.1±0.9 CFU/g was transferred from the chicken contaminated hands to lettuce

58

leaves and 4.3±0.6 to lettuce from the chicken contaminated cutting board (5). In this study,

latex gloves similar in thickness and texture similar to those used in harvest and post-harvest

processing of living lettuce. It is possible that the gloves’ physical texture and design may

improve or inhibit the transfer of bacteria. Additionally, leaks may develop in the gloves,

exposing the lettuce to potential hand contamination, emphasizing the importance of good hand

washing practices. Latex gloves were less frequently associated with leaks compared to vinyl

gloves, another common choice for food service workers (19). Intervention tasks of maintaining

good hygiene by hand washing prior to wearing gloves and changing gloves frequently can

minimize risk of contamination from hands to ready-to eat produce.

This study compared the effectiveness of two leaf selection strategies on the log CFU/g

of Salmonella recovered from lettuce inoculated using worker hands. In this study, only a small

difference of 0.5 log CFU/g could be detected, suggesting current leaf sampling strategies are

adequate to detect worker associated contamination. It was beyond the scope of this study to

examine how the recovery method influenced the detection. Previous studies by Jacobson et al.

(12) compare recovery of Salmonella from three preparation methods: soaking, stomaching, and

blending followed by enrichment according to methods described by FDA’s Bacteriological

Analytical Manual (BAM). Recovery of Salmonella on the samples was influenced by the initial

method used to dissociate the cells from the leaf surface with 344/540 Salmonella positive using

soaked method, 293/540 Salmonella positive using stomaching method, and 232/540 Salmonella

positive using the blending method (12). In contrast, Burnett et al. (3) showed that there was

significant difference in recovery of Salmonella from 26 various types of produce including

fruits, vegetables, and herbs when washing in peptone water, stomaching, or homogenizing were

compared. These differences may reflect particular challenges associated with the

59

morphological characteristics, chemical composition, and pH of leafy greens (3). Further

investigation should examine additional preparation methods for recoverability of Salmonella

isolating from various cultivars and physical characteristic interactions with leafy green leaves.

Physical characteristics such as leaf region, texture and age should be taken into

consideration in screening procedures for detection of contamination of lettuce (2, 14). We

chose mature and intact living lettuce heads for the focus of our study and our samples were

purchased from a local retailer in Blacksburg, VA; therefore we do not know the exact age of the

lettuce at the time of inoculation. The typical shelf life of living lettuce is 2 weeks to 3 weeks

depending on cultivars (18). There are factors that could influence survival and recoverability of

Salmonella from lettuce including age, cultivar, how it was produced and processed, and

handling by distributor and retailers, all of these are unknown in the current study. Brandl et al.

(2) investigated the relationship between ‘Romaine’ leaf age and Escherichia coli O157:H7 and

Salmonella enterica contamination. Both pathogens reached 10-fold higher population sizes on

young leaves compared to middle leaves at pre-harvest and post-harvest stages in the presence of

water on leaves and temperature of 28˚C (2). This suggests young leaves are more likely to

harbor pathogens compared to middle leaves. In addition, nitrogen analysis taken at various leaf

age revealed that young leaves were overall richer in total nitrogen and carbon content

supporting growth of the pathogens on the leaf surface (2). Conversely, Kroupitski et al. (14)

proposes that older ‘Romaine’ leaves were the preferred choice of Salmonella enterica serovar

Typhimurium attachment compared to younger leaves. It was reported that preferred attachments

on surfaces were localized closer to the petiole and on the abaxial side, lower surface of the leaf.

In addition, Scanning electron microscopy demonstrated that surface complexity changes as leaf

ages, indicating the preferred attachment may coordinate with surface structural texture as it ages

60

(14). Kroupitski et al. (15) demonstrated attachment by various Salmonella enterica serovars on

intact and cut lettuce leaves, is improved for biofilm producing strains. This is of particular

concern as biofilms can be present in the pipes and on gutters used for production of hydroponic

lettuce (15).

This study demonstrated a wide dispersal of Glo Germ™ across the whole lettuce head

and leaf’s surface areas; including areas that were not directly contacted by hand were

contaminated possibly through contaminated water droplets (Figure 3.2). It is likely that

contaminated water droplet transfer further spread the contamination. Therefore, simply

removing the outer most leaves may not be the only intervention step to minimized bacterial

contamination. To the best of our knowledge, no data are available concerning the interaction of

Glo Germ™ and Salmonella and it was beyond the scope of this study to examine interaction of

Glo Germ™- Salmonella solution with lettuce tissue. However, the large size of the fluorescent

microsphere particles suggests its behavior would differ compared to a pathogen and is therefore

not a good surrogate for survival and detection. Furthermore, since a reproducible method to

quantify fluorescence of the Glo Germ™ could not be identified this method does not seem

promising for quantification of transfer. Despite use of lecithin as an emulsifier to promote equal

dispersion there were inconsistencies in the amount of fluorescence detected before and after

application of the Glo Germ™ to the leaf (Figure 3.3). Large amounts of variability were

associated with older and outer leaves of same age and region within the leaf. In addition,

increased fluorescence over time was observed that may be related to detection of lysed

chloroplasts being released, resulting in conflicting average quantity readings influencing the

standard curves. The coefficient of determination was =0.68603. Despite, the inability to

quantify transfer in this study Glo Germ™ has the potential to visualize risks of contamination

61

associated with different post-harvest handling practices (6). Vorst et al. (26) have used Glo

Germ™ powder to identify contact surfaces of deli slicers most likely to be contaminated during

slicing, thereby identifying target areas for sanitation (26). Therefore, Glo Germ™ should be

considered as a training tool to inform producers and processors of produce of potential areas

within the environment subject to cross-contamination, that can be targeted for improved

sanitation. Food handler training programs should also consider incorporating Glo Germ™ as

part of training programs to help handlers see a visual demonstration that their actions can lead to

spread of droplets and bacteria that are not visible to the naked eye. The outcome of this training

tool will help workers to understand the important of maintaining good hygiene practices and

sanitation practices.

Preliminary studies were carried out to determine XLT-4 containing rifampicin, an

antibiotic, as a selective agent allowing discrimination of the inoculated strain from native plant

bacteria of the roots and leaves. The ideal media should have no counts when non-inoculated

control roots are plated and high recovery of Salmonella distinctive colonies from inoculated

roots. Results showed successful prevention and XLT-4-Rif selective agar plates were used for

isolation of non-typhi Salmonella colonies for the duration of this study.

CONCLUSION

In conclusion, Glo Germ™ has the potential to be utilized as a teaching aid to promote

safe handling awareness of leafy greens and minimized cross-contamination risks. Our study

proposes that the current FDA methodology of detecting contamination is adequate. However,

risk management and continuing research effort is a must to provide analysis with methods that

are effective for detection and isolating of Salmonella from living lettuce. Appropriate post-

harvest sanitation practices to prevent contamination remains one of the most important

62

measures for ensuring the microbiological safety of living lettuce and the well-being of the

consumers.

63

REFERENCES

1. Blaser, M. J., and L. S. Newman. 1982. A Review of human Salmonellosis: I. infective

dose. Reviews of Infectious Diseases. 4:1096-1106.

2. Brandl, M. T., and R. Amundson. 2008. Leaf age as a risk factor in contamination of

lettuce with Escherichia coli O157:H7 and Salmonella enterica. Applied and Environmental

Microbiology. 74:2298-2306.

3. Burnett, A. B., and L. R. Beuchat. 2001. Comparison of sample preparation methods for

recovering Salmonella from raw fruits, vegetables, and herbs. Journal of Food Protection.

64:1459-1465.

4. Centers of Disease Control and Prevention. 1998. Outbreak of Campylobacter Enteritis

associated with cross-contamination of food-oklahoma, 1996. Available at:

http://www.cdc.gov/mmwr/preview/mmwrhtml/00051427.htm. Accessed July 2012.

5. Chen, Y., K. M. Jackson, F. P. Chea, and D. W. Schaffner. 2001. Quantification and

variability analysis of bacterial cross-contamination rates in common food service tasks. Journal

of Food Protection. 64:72-80.

6. Crandall, P.G., A. Neal, C.A. O’Bryan, C.A. Murphy, B.P. Marks, and S.C. Ricke. 2011.

Minimizing the risk of Listeria monocytogenes in retail delis by developing employee focused,

cost effective training. Agriculture, Food and Analytical Bacteriology. 1:159-174

7. Davis, H., J. P. Taylor, J. N. Perdue, G. N. Stelma, R. Rowntree, and K. D. Greene. 1988.

A Shigellosis outbreak traced to commerically distributed shredded lettuce. American Journal of

Epidemiology. 128:1312-1321.

8. Hammack, T. S., R. M. Amaguana, and W. H. Andrews. 2001. An improved method for

the recovery of Salmonella serovars from orange juice using universal preenrichment broth.

Journal of Food Protection. 64:659-663.

9. Hammack, T. S., I. E. Valentin-Bon, A. P. Jacobson, and W. H. Andrews. 2004. Relative

effectiveness of the bacteriological analytical manual method for the recovery of Salmonella

from whole cantaloupes and cantaloupe rinses with selected preenrichment media and rapid

methods. Journal of Food Protection. 67:870-877.

10. Hammack, T. S., R., Amagua, M. Amaguan, G. A. June, P.S. Sherrod, and W.H.

Andrews. 1999. Relative effectiveness of selenite cystine broth, tetrathionate broth, and

rappaport-vassiliadis medium for the recovery of Salmonella spp. from foods with a low

microbial load. Journal of Food Protection. 62:16-21.

11. Herman, K. M., T.L. Ayers, and M. Lynch. 2008. Foodborne disease outbreaks

associated with leafy greens 1973-2006. International Conference on Emerging Infectious

Disease Poster Abstract.

64

12. Jacobson, A. P., V. S. Gill, K. A. Irvin, H. Wang, and T. S. Hammack. 2012. Evaluation

of methods to prepare samples of leafy green vegetables for preenrichment with the

bacteriological analytical manual Salmonella culture method. Journal of Food Protection.

75:400-404.

13. Kaneko, K.I., H. Hayashidani, K. Takahashi, Y. Shiraki, S. Limawongpranee, and M.

Ogawa. 1999. Bacterial contamination in the environment of food factories processing ready-to-

eat fresh vegetables. Journal of Food Protection. 62:800-804.

14. Kroupitski, Y., R. Pinto, E. Belausov, and S. Sela. 2011. Distribution of Salmonella

Typhimurium in romaine lettuce leaves. Food Microbiology. 28:990-997.

15. Kroupitski, Y., R. Pinto, M. T. Brandl, E. Belausov, and S. Sela. 2009. Interactions of

Salmonella enterica with lettuce leaves. Journal of Applied Microbiology. 106:1876-1885.

16. Montville, R., Y. Chen, and D. W. Schaffner. 2001. Glove barriers to bacterial cross-

contamination between hands to food. Journal of Food Protection. 64:845-849.

17. Moore, C. M., B. W. Sheldon, and L. A. Jaykus. 2003. Transfer of Salmonella and

Campylobacter from stainless steel to romaine lettuce. Journal of Food Protection. 66:2231-

2236.

18. Morgan, L. 1999. Hydroponic lettuce production a comprehensive, practical and

scientific guide to commerical hydroponic lettuce production. Casper publications pty ltd

narrabeen, new zealand.

19. Olsen, R. J., P. Lynch, M.B. Coyle, J. Cummings, T. Bokete, and W. E. Stamm.1993.

Examination gloves as barriers to hand contamination in clinical practice. Journal of the

American Medical Assoication. 270:350-353.

20. Painter, J.A., T. Ayers, R.V. Tauxe, C.R. Braden, F.J. Angulo et al. 2013. Attribution of

foodborne illnesses, hospitalizations, and deaths to food commodities by using outbreak data,

united states, 1998–2008. Emerging Infectious Diseases. 19.

21. Stafford, R. J., B. J. McCall, A.S. Neill, D. S. Leon, G. J. Dorricott, C.D. Towner, and

G.R. Micalizz. 2002. A statewide outbreak of Salmonella Bovismorbificans phage type 32

infection in Queenland. Communicable Diseases Intelligence Quartely Report. 26:568-573.

22. Strayer, R.F. 1994. Dynamics of microorganism populations in recirculating nutrient

solutions. Advances in Space Research. 14:357-366.

23. Sumathi, S., R. F. Cindy, C. Linda, and V. T. Robert. 2004. Fresh produce: A growing

cause of outbreaks of foodborne illness in the United States, 1973 through 1997. Journal of Food

Protection. 67:2342-2353.

65

24. Taormina, P.J., L. R. Beuchat, M. C. Erickson, L. Ma, G. Zhang, and M. P. Doyle. 2009.

Transfer of Escherichia coli O157:H7 to iceberg lettuce via simulated field coring. Journal of

Food Protection. 72:465-472.

25. U.S. Food and Drug Administration. 2011. FDA’s bacteriological analytical manual

(BAM) chapter 5: Salmonella. Available at:

http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManua

lBAM/ucm070149.htm. Accessed September 2012.

26. Vorst, K. L., E. C. D. Todd, and E. T. Ryser. 2006. Transfer of Listeria monocytogenes

during mechanical slicing of turkey breast, bologna, and salami. Journal of Food Protection.

69:619-626.

27. Wachtel, M. R., and A. O. Charkowski. 2002. Cross-contamination of lettuce with

Escherichia coli O157:H7. Journal of Food Protection. 65:465-470.

66

FIGURES

Figure 3.1: Red food coloring was used as an indicator to visualize where the gloves came into

contact with the different parts of lettuce plants during routine packaging of living lettuce.

Figure 3.2: Droplets of Glo Germ™ solution on a lettuce head visualized using blacklight.

A) Living lettuce viewed on its side, showing the distribution from bottom to top of the leaf.

B) View of top of the head of living lettuce, showing the wide dispersion throughout the whole

surface area and inner leaves.

67

Figure 3.3: Images of older and outer lettuce leaves used to calculate the average intensity

(Average Quantity) within a 30x30 mm area. The average quantity value of the leaf without

(background) Glo Germ™ was subtracted from the average intensity value of the leaf with Glo

Germ™ to determine the fluorescence only by Glo Germ™.

68

Figure 3.4: Enumeration of Salmonella Enteritidis ptvs 177 recovered from living lettuce leaves

chosen using two different selection methods: random selection and blacklight to visualize Glo-

Germ co-inoculated with the Salmonella.

Data shows the average log CFU/g of Salmonella and error bars from 3 replicates (n=9) and cells

were recovered by plating on XLT4-Rif and incubated at 37˚C for 48 h. Each error bar is

constructed using 1 standard deviation from the mean. Samples not connected by same letter are

significantly different (P<0.05)

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

Random Blacklight

log C

FU

/g

Selection methods

A

B

69

Figure 3.5: The recoverability of Salmonella Enteritidis ptvs 177 following a single

contamination event from worker gloves followed by sequential post-harvest packing of living

lettuce using two different selection methods.

Data shows the average log CFU/g of Salmonella and error bars from 3 replicates (n=9) and cells

were recovered by plating on XLT4-Rif and incubated at 37˚C for 48 h. Each error bar is

constructed using 1 standard deviation from the mean. Samples not connected by same letter are

significantly different (P<0.05)

c

A

70

CHAPTER 4: POST-HARVEST TRANSFER AND SURVIVAL OF Salmonella enterica

SEROTYPE ENTERITIDIS ON LIVING LETTUCE

ABSTRACT

A number of risk factors for post-harvest contamination of field-harvested lettuce have

been described; however, the potential for post-harvest transfer of Salmonella in small-scale

hydroponic systems is not well understood. The purpose of our study was to quantify the transfer

and survival of Salmonella enterica Enteritidis from contaminated roots to the leaves of lettuce

packaged as “living lettuce” and stored at 4˚C and 12˚C. Living lettuce is packaged within

clamshell with intact roots, greatly extending shelf life to 18 days. Butterhead lettuce cultivar

‘Buttercrunch’ was grown hydroponically, harvested, and roots were inoculated with Salmonella

by soaking. The roots were wrapped in a knot and the lettuce placed in clamshells stored at

recommended temperature (4˚C) or under temperature abuse conditions (12˚C). Periodically

three packages were removed and lettuce destructively processed. Roots and leaves were

removed from the head, homogenized in peptone water separately and Salmonella enumerated

by serial dilution and plating onto XLT-4 agar. On average 5.1±0.00 log CFU/g of Salmonella

were transferred to the roots from inoculated solution, while only 2.9±0.1 log CFU/g Salmonella

were transferred to leaves from inoculated roots. Salmonella persisted but did not grow on

leaves when stored at 4˚C or 12˚C for 18-days. Storage at 12˚C was associated with 2 log CFU/g

increases in Salmonella on roots after 18-days storage (P=0.0002), while 4˚C storage was

associated with a decrease of 0.4 log CFU/g Salmonella on roots (P=0.0001). This reinforces the

need for maintaining temperature control and identifying risks associated with post-harvest

handling and distribution.

71

INTRODUCTION

In the United States, outbreaks of food-borne illnesses are increasingly attributed to fresh

produce, with an estimated 46% of food-borne illnesses attributable to produce (25). Produce-

associated contamination accounted for 190 outbreaks including 16,058 illnesses, 598

hospitalizations, and 8 deaths between 1973 and 1997 (30). The public health burden associated

with contaminated leafy greens has continued to grow with a 38% increase in leafy greens

outbreaks from 1996-2005 (10). Between 1998-2008, outbreaks attributable to leafy greens were

associated with 186,140 illnesses, 2,367 hospitalizations, and 26 deaths (25). These produce

associated illnesses and outbreaks create an annual economic burden of $39 billion (27).

Salmonella enterica was the etiologic agent in 35/502 leafy greens outbreaks between 1973-2006

in the United States (10). Internationally, outbreaks of Salmonella Thompson (23) and

Salmonella Braenderup (7) were traced back to contaminated lettuce. Contamination of lettuce

and leafy greens can occur anywhere along the farm to fork continuum. Risk prevention and

management are critical factors to maintain produce safety and economical value of the food

industry (1).

There has been an increase interest in development and implementation of Good

Agricultural Practices (GAPs) to reduce produce contamination. GAPs guidance documents are

available for field-grown leafy greens but does not address hydroponically produced living

lettuce. In the United States, living lettuce with intact roots packaged in plastic clamshells is

increasing in popularity. Living lettuce is typically produced in environmentally controlled

greenhouses using hydroponic systems. Hydroponic systems incorporate the sustainable

agricultural practice of growing produce in mineral supplemented water without soil (9). One

attractive benefit of hydroponics systems is the short reproduction cycle within crops that

72

generate high yields; this makes them ideal candidates for soilless systems, which offer plant

nutrients precision that could increase the yields of leafy greens (6, 22). Pre-harvest

contamination by insects, wildlife and run-off is reduced in greenhouse hydroponic systems but

other sources of contamination remain, chiefly water used for irrigation, foliar application. Post-

harvest contamination during harvesting, packaging and washing are associated with increased

spread of contamination (30, 38). Water is a recognized vehicle that allows pathogens to be

distributed to produce. Studies have demonstrated potential for transfer from water used for

overhead irrigation to edible tissue (11, 13, 18, 29, 33). Koseki et al. (17) recently identified

much greater amounts of transfer of human pathogens to the roots of hydroponically grown

spinach plants compared to the leaves (17). This suggests the primary route of contamination in

the hydroponic system is through the roots rather than direct application of pathogen

contamination onto the surface of the leaves (17). Therefore, handling practices for living lettuce

are likely to transfer pathogens from contaminated roots to the edible tissue during packaging.

Yet, the survival on intact roots and potential for transfer to edible leaves of living lettuce is

poorly understood.

The objective of our study was to quantify the transference of Salmonella enterica

serotype Enteritidis from contaminated roots to the leaves of a mature Butterhead lettuce

packaged as “living lettuce” in a clamshell with intact roots. In addition, the survival of

Salmonella on lettuce roots and leaves stored at typical retail storage conditions of 4˚C and

temperature abuse conditions of 12˚C was determined throughout shelf life. These research

findings will provide knowledge about cross contamination in post-harvest hydroponic systems

and promote implementation of safe handling strategies and risk management.

73

MATERIALS AND METHODS

Bacterial strain and culture conditions.

The Salmonella enterica Enteritidis strain ptvs177 obtained from Trevor Suslow was

passed through increasing concentrations of rifampicin (Fisher Scientific, Fair Lawn, NJ) until

resistance to concentrations of 100 µg/ml was achieved. The culture was maintained in Tryptic

Soy broth (TSB; Difco, Sparks, MD) supplemented with 100 µg/ml rifampicin (TSB-Rif; Fisher

Scientific) and 30% Glycerol (Fisher Scientific) and stored at -80˚C. Bacterial cultures were

prepared by sub-culturing from a frozen stock in TSB-Rif and incubated statically at 37˚C for 24

h. Colony morphology and selective indicators were confirmed by plating onto Xylose-Lysine-

Tergitol-4-Agar (XLT-4; Difco) containing 100 µg/ml rifampicin (XLT-4- Rif) and incubated

statically at 37˚C for 24 h. Positive colonies for Salmonella spp. were red with black centers.

Greenhouse growing conditions, harvest, and storage conditions of living lettuce.

Butterhead lettuce cultivar ‘Buttercrunch’ was grown in hydroponic system to maturity at

the Virginia Tech Aquaculture Research Center, Saltville, VA. Seeds were germinated in perlite

and transplanted into continuous-flow Nutrient Film Technology (NFT) channels at the 2-leaf

stage. Lettuce heads were harvested at maturity after 48 days after transplantation to NFT for

trial 1 (n=39) and 58 days for trial 2 (n=39). Differences in times to harvest were associated with

the decreased day length associated with the second trial. Lettuce with intact root systems were

harvested following typical living lettuce handling practices: 1) lift a head of lettuce by the base

using gloved hands, 2) separate roots from neighbor leaving at least 2 inches of roots on either

side, 3) squeeze excess water from the lettuce roots, 4) lettuce head with intact roots was

weighed, 5) wrap the lettuce roots into a knot form, and 6) transfer the lettuce to a plastic lettuce

clamshell (ProducePackaging.com). The living lettuce clamshells were transported on ice to the

74

Food Science and Technology building at Virginia Tech, and then stored overnight at 4˚C to

remove greenhouse heat.

Inoculation of lettuce roots, post-harvest handling, and storage conditions of living lettuce.

The follow morning after harvest (12-16 h), lettuce roots were inoculated with

Salmonella enterica Enteritidis by immersion for 10 min in nutrient solution within a sterile 500

ml Erlenmeyer glass flask. (Figure 4.1). The nutrient solution contained 1.0 ml of Salmonella

culture grown statically 24 h in TSB-Rif (OD 580nm = 0.8) in 400 ml 0.1% sterile buffer peptone

water (BPW; Sigma- Aldrich). To prevent contamination of leaves during root submersion the

lettuce head was encased in a Ziploc® double zipper bag (26.8cm x 27.3cm) with a small hole

allowing only the roots to protrude. Handling practices were performed as follows: 1) lift a head

of lettuce by the base using gloved hands, 2) the roots were squeezed to remove excess water, 3)

wrap lettuce roots forming a knot, and 4) immediately transfer the lettuce with wrapped roots to

clamshell container (Figure 4.1).

To examine post-harvest survival of Salmonella, two different storage temperature

conditions were selected for this study. In trial 1, lettuce packaged in plastic, clear clamshells

was stored at 12˚C to simulate temperature abuse conditions that may occur in a holding facility,

or in a consumer’s kitchen. In trial 2, lettuce was held at 4˚C, the FDA recommended

temperature for storage of minimally processed produce (35). The study was terminated when

signs of reduced produce quality (loss of sample color, loss of cell turgor, tissue weeping and the

development of sliminess) were observed.

Microbiological sampling and enrichment.

Lettuce samples were processed on days 0, 1, 2, 3, 6, 9, 12, 15, and 18 to enumerate the

survival of Salmonella CFU/g stored at 4˚C or 12˚C. For each sampling day, three randomly

75

selected living lettuce clamshells were removed from the designated storage. The roots were

removed at the base/root interface using an ethanol flamed knife and a cutting board covered

with disposable cutting sheets. The knife was flame sterilized between each head and new

cutting sheets used. Ten grams of root (f.w.) were transferred to a filter bag, immersed in 90 ml

of 0.1% BPW, and the bag contents were homogenized for 2 minutes in a lab blender (Bag

Mixer, Interscience, Weymouth, MA). Leaves were randomly removed from lettuce heads and

25 g transferred to a filter bag. Leaves were homogenized for 2 minutes in 225 ml of BPW

containing 0.1% Tween 80 (Fisher Scientific) as described in FDA’s (U S Food and Drug

Administration) Bacteriological Analytical Manual (BAM) (36). Enumeration of Salmonella

were performed using serial dilution into BPW and subsequent spread plating onto XLT-4-Rif.

Numbers of Salmonella colonies after 48 hours of incubation at 37˚C were recorded.

Additionally, 1.0 ml of homogenized contents (leaves) from stomacher bag was added to 9 ml of

TSB supplemented with rifampicin and incubated at 37˚C for 24 h. After 24 h the culture was

streaked for isolation on XLT-4-Rif and incubated at 37˚C for 24 h. Non-inoculated lettuce

controls (n=3) were processed as above to account for contamination or growth of non-

Salmonella bacteria on the selective media.

Statistical analysis.

Bacterial counts (CFU/g) were log transformed to approximate normal distribution. Data

was subjected to one-way ANOVA using statistical software JMP® Pro 10.0 (SAS; Institute

Inc., Cary, NC). The post-harvest survival of Salmonella log CFU/g averages recovered from

two different temperatures, 4˚C and 12˚C, using 39 heads of living lettuce at each trial was

statistically investigated by least significant differences (LSD), to denote differences. P-values

(P< 0.05) with =0.5 being considered significant.

76

RESULTS

Shelf life study of living lettuce.

The shelf life of living lettuce in this study was 18-days, regardless of storage

temperature. After this point no further enumeration of Salmonella was performed.

Post-harvest transfer and survival of Salmonella Enteritidis on living lettuce.

On average 5.1±0.0 log CFU/g of Salmonella was transferred to the roots from the

nutrient solution and 2.9±0.1 log CFU/g was detected on the leaves. At 4˚C and 12˚C,

Salmonella cells persisted on the edible leaves for the 18-days of the study (Figure 4.2 and 4.3).

Storage at 12˚C was associated with an increase of 1.6 log CFU/g Salmonella on roots of living

lettuce after 18-days (P=0.0002). Salmonella also increased by 0.62 log CFU/g Salmonella on

living lettuce leaves (P=0.0017) when stored at 12˚C for 18-days (Figure 4.2). No increases in

log CFU/g of Salmonella occurred on living lettuce stored at 4˚C for 18-days (Figure 4.3). After

6 days of storage at 4˚C Salmonella decreased by 0.4 log CFU/g on roots (Figure 4.3), but

remained unchanged throughout the remainder of the 18-day period (P<0.0001). Non-inoculated

controls (n=3) yield no counts.

DISCUSSION

Post-harvest transfer of Salmonella and other human pathogens to produce may be

associated with increased risk of transmission to humans and increased incidence of enteric

disease. Post-harvest handling marks the beginning of physiological changes by cutting,

trimming, and washing. During these steps post-harvest contamination spread can occur through

contaminated water, via infected handler, contact surface, and knives (5, 26). In this study, direct

immersion of the roots in a low-nutrient solution containing Salmonella was used to simulate an

event where the irrigation water used for hydroponic production was contaminated.

77

Contamination of lettuce and spinach roots has been demonstrated previously (14, 15, 28).

Salmonella serovars Typhimurium, Dublin, Cancan, Nelly, and Tamburo were able to internalize

into lettuce seedlings grown in 0.5% Hoagland’s agar (14, 15). These studies feature inclusion of

agar to the nutrient solution, which may increase interaction of motile cells with root hairs and

facilitate uptake or attachment (14, 28). It was beyond the scope of the current study to determine

if Salmonella enterica were internalized through the roots and travelled through the roots to the

leaf surface. Prior studies propose evidence of Salmonella enterica internalization (8, 16, 29, 31).

However, this study did recover Salmonella from the leaf surfaces, when only the roots were

inoculated. Contamination of human pathogens from gloves and utensils have been

demonstrated, with as much 2 log CFU/g from hands to lettuce leaves (4). In this study, care

was taken to assure that the leaves did not come into direct contact with the nutrient solution at

the time of inoculation. Plastic bags were used to encase lettuce heads to prevent any transfer of

Salmonella from handler’s hands to the leaves during handling. The bags were cut from the

lettuce heads after the head was placed within the clamshell to prevent further handler associated

transfer. It is likely that water droplets from the roots were transferred to the plastic clamshell

and then to the edible tissue during transport in and out of incubators used for storage. The

interaction of pathogen survival linked to packaging material is beyond the scope of this

research. As the handling steps practiced in this study were similar to those practiced by small

hydroponics producers, the risk associated with post-harvest contamination of living lettuce is

likely underestimated. Future studies should examine additional strategies to reduce humidity

within clamshells and prevent droplet transfer.

Persistence of Salmonella and other human pathogens on fresh, minimally processed

produce is dependent, at least in part on storage temperatures. In this study Salmonella persisted

78

at 4˚C over the 18-day shelf life. Our results indicated a decrease of 0.4 log CFU/g Salmonella

recovered on roots after 6 days of storage at 4˚C( P<0.0001). Our results were comparable to Hsu

et al. (12), first 5 days of storage at 4˚C a significantly decrease of 0.47 to 0.8 log CFU/g

Salmonella on basil, parsley, rosemary, and cilantro was reported. Although Wu et al. (39)

reported Shigella sonnei reduced by 2.5 to 3.0 log CFU/g on inoculated parsley leaves stored at

4˚C for 14 days. Tian et al. (32) reported no significant differences were observed in the growth

of 4 pathogens on minimally processed vegetables stored at 4˚C for 15 days. It was observed

E. coli O157:H7 and Salmonella Typhimurium increased by 2 log CFU/g when stored at 15˚C

for 1 day. Results concluded survival and growth of pathogens were impacted by storage

temperature and time (32).

Pathogens on contaminated produce can grow more rapidly once chopped, releasing

nutrients and water for pathogen growth held at room temperature (26). Ukuku et al. (34)

evaluated the effects of storage temperature abuse on Salmonella linked to fresh cut watermelon

stored at 5˚C and 10˚C for 12 days. Their findings were at 5˚C Salmonella declined by 1 log

CFU/g; yet for 10˚C Salmonella increased by 2 to 3 log CFU/g (P<0.05). This finding was

consistent with our results, where the trimming of roots may have released nutrients allowing for

the growth observed at 12˚C . Luo et al. (19) observed 2 log CFU/g statistical increase in

Escherichia coli O157:H7 on packaged fresh-cut salads containing ‘Romaine’ and ‘Iceberg’

lettuce when held at 12˚C. Limited growth was recorded when lettuce was held at 5˚C, in

agreement with our results. Oliveira et al. (24) report increases of 2.4 and 4.2 log CFU/g of

Salmonella enterica and Escherichia coli respectively when inoculated onto shredded ‘Romaine’

lettuce and stored in modified atmosphere packaging (MAP) at 25˚C for 3 days. These

observations are significant suggesting that both Escherichia coli O157:H7 and Salmonella can

79

grow, achieving amounts comparable to the average infectious dose, on packaged fresh-cut

lettuce while visual quality is acceptable to consumers. This creates concerns as the potential risk

of amplification of pathogens increases when living lettuce is stored at temperature abusive

conditions. The interaction of pathogen survival linked to packaging material is beyond the scope

of this research. Further research is necessary to conclude the survival of pathogens associated

with packaging clamshells and refrigerated produce. Small scale hydroponic producers typically

distribute living lettuce to farmers’ markets where temperature control may be difficult to

maintain below 12˚C; consumers face a significant risk in purchasing these contaminated lettuce

heads. Pathogens in contaminated living lettuce have the potential to amplify and persist at

temperature abusive conditions. Food safety surveys conducted in Europe, North America, and

Australia indicate that consumers have knowledge gaps in refrigeration practices (26). The

surveys revealed up to 93% of consumers were unaware that to prevent growth of the majority of

human pathogens refrigeration temperature is 0˚-5˚C (26). In United States, 40-56% of the

population demonstrated incorrect refrigeration temperature practices (26). Results concluded

from surveys conducted in Sweden that 5-20% of food items were stored at temperatures above

10˚C; majority of food items stored at temperatures ranged from 11.3-13.6 ˚C (20). The largest

percentage of samples (19%) held above 10˚C were Ready-To-Eat (R-T-E) salads with the

maximum temperature recorded 18.2˚C (20). The results of this study highlight the need to store

living lettuce at 4˚C immediately after harvest to improve the microbiological safety.

Refrigeration at proper temperature will prevent amplification of harmful bacteria pathogens on

produce. Temperature abuse is the main source of microbial and quality deterioration in produce

(5). Fluctuations in temperature (<10˚C) can occur during improper storage-holding conditions,

shipping and unloading of fresh-cut produce at the distributors (5). Our samples were

80

transported on ice and maintained <10˚C in coolers to minimize fluctuations during

transportation and storage to optimized shelf life quality. According to the FDA Model Food

Code 2009 guidelines, produce must be received and stored at 5˚C (2). This reinforces the need

for maintaining temperature control to reduce bacterial pathogen contamination.

Our lettuce harvest for trial 2 study held at 12˚C had algae present on the roots. Although

algae were carefully removed there was still some residue on the roots. It is unknown if the

presence of algae on the roots improved or inhibited the post-harvest survival of Salmonella. The

presence of algae can impact food safety by damaging lettuce roots or limiting valuable nutrients

for growth use. Research findings on effective algae control and disinfection agents to improve

living lettuce safety produced in a environmental controlled greenhouse. Further research is

necessary to determine if the presence of algae improves the survival of harmful human

pathogens through root attachment.

Preliminary studies were carried out to determine XLT-4 containing rifampicin, an

antibiotic, as a selective agent allowing discrimination of the inoculated strain from native plant

bacteria of the roots and leaves. The ideal media should have no counts when non-inoculated

control roots are plated and high recovery of Salmonella distinctive colonies from inoculated

roots. Results showed successful prevention and XLT-4-Rif selective agar plates were used for

isolation of non-typhi Salmonella colonies for the duration of this study. Our study was

performed to determine the best optimum time for soaking lettuce roots to ensure Salmonella

recoverability. Roots were soaked in nutrient solution at variable times: 10 min, 30 min, and 60

min. There was no statistical difference in numbers of Salmonella recovered with the different

inoculation times; therefore to duration of the studies roots were soaked in nutrient solution for

10 min.

81

CONCLUSION

In conclusion, the results of our study revealed that direct contamination of Salmonella

Enteritidis on the roots of living lettuce transferred to the edible leaves; remained on roots and

grew as temperature abuse conditions increased. Maintaining temperature control and risks

associated with contamination in hydroponic production is emphasized by these important

findings. To the best of our knowledge small scale hydroponic producers are not required by law

to follow recognized safe post-harvest handling practices. A recognized food safety guideline by

the FDA, “Guide to Minimize Microbial Food Safety Hazards for Fresh Fruits and Vegetables”,

has recommendations on food safety handling practices for field production of vegetable crops;

however, it has not addressed food safety related to soilless vegetable crops (37).

Implementations of food safety guidelines coupled with risk preventions that are practical to

small scale hydroponic producers are important. Application of GAPs (Good Agricultural

Practices) and GHPs (Good Handling Practices) are suggested to increase food safety by

minimizing pathogen contamination during post-harvest handling. This creates awareness on

contamination pathways and how they may be prevented; establishing effective management

practices (2, 30, 35, 37). Limit resources on produce consumption without cooking to reduce

illnesses are available to consumers (2, 30, 35, 37). FDA recommends washing produce,

removing bruised or damaged areas immediately before eating (2, 30, 35, 37). Beuchat et al. (3)

demonstrated that produce washed with potable water removes loose dirt and reduces pathogen

load by 1 to 2 log CFU/g, but does not completely eliminate it. Therefore, reinforcing the

importance of strict hygiene practices during harvesting and packaging is essential to maintain

food safety.

82

REFERENCES

1. Ahmer, B. M. 2004. Cell-to-cell signalling in Escherichia coli and Salmonella enterica.

Mol Microbiol. 52:933-45.

2. Anonymous. 2009. Food Code 2009 U. S. Department of Health and Human Services

Available at:

http://www.fda.gov/Food/FoodSafety/RetailFoodProtection/FoodCode/FoodCode2009/ .

Accessed February 2013.

3. Beuchat, L. R. 1996. Pathogenic microorganisms associated with fresh produce. Journal

of Food Protection. 59:204-216.

4. Chen, Y., K. M. Jackson, F. P. Chea, and D. W. Schaffner. 2001. Quantification and

variability analysis of bacterial cross-contamination rates in common food service tasks. Journal

of Food Protection. 64:72-80.

5. Delaquis, P., S. Bach, and L.D. Dinu. 2007. Behavior of Escherichia coli O157:H7 in

leafy vegetables. Journal of Food Protection. 70:1966-1974.

6. Fallovo, C., Y. Rouphael, E. Rea, A. Battistelli, and G. Colla. 2009. Nutrient solution

concentration and growing season affect yield and quality of Lactuca sativa l. var. acephala in

floating raft culture. Journal of the Science of Food and Agriculture. 89:1682-1689.

7. Gajraj, R., S. Pooransingh, J. I. Hawker, and B. Olowokure. 2011. Multiple outbreaks of

Salmonella Braenderup associated with consumption of iceberg lettuce. International Journal of

Environmental Health Research. 22:150-155.

8. Golberg, D., Y. Kroupitski, E. Belausov, R. Pinto, and S. Sela. 2011. Salmonella

Typhimurium internalization is variable in leafy vegetables and fresh herbs. International

Journal of Food Microbiology. 145:250-257.

9. Grewal, H. S., B. Maheshwari, and S. E. Parks. 2011. Water and nutrient use efficiency

of a low-cost hydroponic greenhouse for a cucumber crop: An Australian case study.

Agricultural Water Management. 98:841-846.

10. Herman, K. M., T.L. Ayers, and M. Lynch. 2008. Foodborne disease outbreaks

associated with leafy greens 1973-2006. International Conference on Emerging Infectious

Disease.

11. Holvoet, K., L. Jacxsens, I. Sampers, and M. Uyttendaele. 2012. Insight into the

Prevalence and distribution of microbial contamination to evaluate water management in the

fresh produce processing industry. Journal of Food Protection. 75:671-681.

83

12. Hsu, W.Y., A. Simonne, and P. Jitareerat. 2006. Fates of seeded Escherichia coli

O157:H7 and Salmonella on selected fresh culinary herbs during refrigerated storage. Journal of

Food Protection. 69:1997-2001.

13. Islam, M., J. Morgan, M. Doyle, S. Phatak, P. Millner, and X. Jiang. 2004. Persistence of

Salmonella enterica serovar Typhimurium on lettuce and parsley and in soils on which they were

grown in fields treated with contaminated manure composts or irrigation water. Foodborne

Pathogens and Diseases. 1:27-35.

14. Klerks, M. M., E. Franz, M. van Gent-Pelzer, C. Zijlstra, and A. H. C. van Bruggen.

2007. Differential interaction of Salmonella enterica serovars with lettuce cultivars and plant-

microbe factors influencing the colonization efficiency. Internaitonal Society Microbial Ecology

Journal. 1:620-631.

15. Klerks, M. M., M. van Gent-Pelzer, E. Franz, C. Zijlstra, and A. H. C. van Bruggen.

2007. Physiological and molecular responses of lactuca sativa to colonization by Salmonella

enterica serovar Dublin. Applied and Environmental Microbiology. 73:4905-4914.

16. Kroupitski, Y., D. Golberg, E. Belausov, R. Pinto, D. Swartzberg, D. Granot, and S. Sela.

2009. Internalization of Salmonella enterica in leaves is induced by light and involves

chemotaxis and penetration through open stomata. Applied and Environmental Microbiology.

75:6076-6086.

17. Koseki, S., Y. Mizuno, and K. Yamamoto. 2011. Comparison of two possible routes of

pathogen contamination of spinach leaves in a hydroponic cultivation system. Journal of Food

Protection. 74:1536-1542.

18. Lapidotand, A., and S. Yaron. 2009. Transfer of Salmonella enterica Serovar

Typhimurium from contaminated irrigation water to parsley is dependent on curli and cellulose,

the biofilm matrix components. Journal of Food Protection. 72:618-623.

19 . Luo, Y., Q. He, and J. L. McEvoy. 2010. Effect of storage temperature and duration on

the behavior of Escherichia coli O157:H7 on packaged fresh-cut salad containing romaine and

iceberg lettuce. Journal of Food Science. 75:M390-M397.

20. Marklinder, I. M., M. Lindblad, L. M. Eriksson, A. M. Finnson, and R. Lindqvist. 2004.

Home storage temperatures and consumer handling of refrigerated foods in sweden. Journal of

Food Protection. 67:2570-2577.

21. Mercanoglu Taban, B., and A. K. Halkman. 2011. Do leafy green vegetables and their

ready-to-eat [RTE] salads carry a risk of foodborne pathogens? Anaerobe. 17:286-287.

22. Nicola, S., J. Hoeberechts, and E. Fontana. 2005. Comparsion between traditional and

soilless culture systems to produce rocket (eruca sativa) with low nitrate concent ISHA Acta

Horticulturae. 697:549-555.

84

23. Nygård, K., J. Lassen, L. Vold, Y. Andersson, I. Fisher, S. Löfdahl, J. Threlfall, I. Luzzi,

T. Peters, M. Hampton, M. Torpdahl, G. Kapperud, and P. Aavitsland. 2008. Outbreak of

Salmonella Thompson infections linked to imported Rucola Lettuce. Foodborne Pathogens and

Diseases. 5:165-173.

24. Oliveira, M., J. Usall, C. Solsona, I. Alegre, I. Viñas, and M. Abadias. 2010. Effects of

packaging type and storage temperature on the growth of foodborne pathogens on shredded

romaine lettuce. Food Microbiology. 27:375-380.

25. Painter J.A., R.M. Hoekstra, T. Ayers, R. V. Tauxe, C. R. Braden, F. J. Angulo et al.

2013. Attribution of foodborne illnesses, hospitalizations, and deaths to food commodities by

using outbreak data, united states, 1998–2008. Emerging Infectious Diseases. 19.

26. Redmond, E. C., and C. J. Griffith. 2003. Consumer food handling in the home: A review

of food safety studies. Journal of Food Protection. 66:130-161.

27. Scharff, R. L. 2010. Health-related costs from food-borne illness in the united states. The

produce safety project at georgetown university. www.producesafetyproject.org. Accessed July

2012.

28. Sharma, M., D. T. Ingram, J. R. Patel, P. D. Millner, X. Wang, A. E. Hull, and M. S.

Donnenberg. 2009. A novel approach to investigate the uptake and internalization of Escherichia

coli O157:H7 in spinach cultivated in soil and hydroponic medium. Journal of Food Protection.

72:1513-1520.

29. Solomon, E. B., S. Yaron, and K. R. Matthews. 2002. Transmission of Escherichia coli

O157:H7 from contaminated manure and irrigation water to lettuce plant tissue and its

subsequent internalization. Applied and Environmental Microbiology. 68:397-400.

30. Sumathi, S., R. F. Cindy, C. Linda, and V. T. Robert. 2004. Fresh produce: a growing

cause of outbreaks of foodborne illness in the united states, 1973 through 1997. Journal of Food

Protection. 67:2342-2353.

31. Teplitski, M., J. D. Barak, and K. R. Schneider. 2009. Human enteric pathogens in

produce: un-answered ecological questions with direct implications for food safety. Current

Opinion in Biotechnology. 20:166-171.

32. Tian, J.Q., Y.-M. Bae, N.Y. Choi, D.-H. Kang, S. Heu, and S.Y. Lee. 2012. Survival and

growth of foodborne pathogens in minimally processed vegetables at 4 and 15 °C. Journal of

Food Science. 77:M48-M50.

33. Tyrrel, S. F., J. W. Knox, and E. K. Weatherhead. 2006. Microbiological water quality

requirements for salad irrigation in the United Kingdom. Journal of Food Protection. 69:2029-

2035.

85

34. Ukuku, D. O., and G. M. Sapers. 2007. Effect of time before storage and storage

temperature on survival of Salmonella inoculated on fresh-cut melons. Food Microbiology.

24:288-295.

35. U.S. Food and Drug Administration. 2006. Commodity specific food safety guidelines

for the lettuce and leafy greens supply chain. Available at:

http://www.fda.gov/downloads/Food/FoodSafety/ProductSpecificInformation/FruitsVegetablesJ

uices/GuidanceComplianceRegulatoryInformation/UCM169008.pdf. Accessed September 2012.

36. U.S. Food and Drug Administration. 2011. FDA’s bacteriological analytical manual

(BAM) chapter 5: Salmonella. Available at:

http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManua

lBAM/ucm070149.htm . Accessed September 2012.

37. U.S. Food and Drug Administration. 2008. Guidance for industry: guide to minimize

microbial food safety hazards of fresh-cut fruits and vegetables. Available at:

http://www.fda.gov/food/guidancecomplianceregulatoryinformation/guidancedocuments/produce

andplanproducts/ucm064458.htm. Accessed September 2012.

38. Wachtel, M. R., and A. O. Charkowski. 2002. Cross-contamination of lettuce with

Escherichia coli O157:H7. Journal of Food Protection. 65:465-470.

39. Wu, F. M., M. P. Doyle, L. R. Beuchat, J. G. Wells, E. D. Mintz, and B. Swaminathan.

Fate of Shigella sonnei on parsley and methods of disinfection. Journal of Food Protection.

63:568-572.

86

FIGURES

Figure 4.1: A series of pictures demonstrating post-harvest handling and inoculation of the

heads. A) Lettuce heads and intact root systems were harvested at Virginia Tech Aquaculture

Research Center Saltville, VA. B) Post-harvest Living lettuce. C) The packaged clamshells

containing living lettuce with intact roots were transported on ice. D) To prevent contamination

of leaves by splashing a Ziploc® plastic bag was used to encase the head with only a small hole

to allow roots to protrude. E) The roots soaking in the nutrient solution containing Salmonella for

10 minutes. F) Wrapped roots forming a knot. G) The knot formed. H) Immediately transfer the

lettuce with wrapped roots to clamshell container. I) Store the packaged lettuce in 4˚C or 12˚C,

respectively, to be processed on sampling days.

87

Figure 4.2. Enumeration of Salmonella recovered from lettuce roots and leaves of living lettuce

held at 12˚C throughout the 18-day shelf life.

Bars reflect the average numbers of log CFU/g from 3 replicates destructively processed per

sampling day recovered by plating on XLT4-Rif and incubated at 37˚C for 48 h. Each error bar is

constructed using 1 standard deviation from the mean. Samples not connected by same letter are

significantly different (P<0.05).

0

1

2

3

4

5

6

7

Day 0 Day 1 Day 2 Day 3 Day 6 Day 9 Day 12 Day 15 Day 18

Log

CFU

/g

Roots

Leaves

E D E

C D E D E

B C B C D B C D A B

A

h g h g h

h g h h g h

f g f

88

Figure 4.3: Enumeration of Salmonella recovered from living lettuce roots and leaves held at

4˚C throughout the 18-day shelf life.

Bars reflect the average numbers of log CFU/g from 3 replicates destructively processed per

sampling day recovered by plating on XLT4-Rif and incubated at 37˚C for 48 h. Each error bar is

constructed using 1 standard deviation from the mean. Samples not connected by same letter are

significantly different (P<0.05).

A

89

CHAPTER 5: CONCLUSION AND FUTURE RESEARCH DIRECTION

The purpose of this work was to study the survival of Salmonella enterica serotype

Enteritidis on roots and leaves of lettuce produced in a controlled greenhouse environment,

packaged with intact roots in a plastic clamshell and stored at 4˚C and 12˚C for an 18-day shelf

life. It was demonstrated through this study, that Salmonella persisted on the roots and leaves of

living lettuce inoculated post-harvest when stored at 4˚C or 12˚C. This creates concerns as the

potential risk of amplification of pathogens increases when living lettuce is stored at high

temperatures. In addition, Glo Germ™ solution was used in the presence of Salmonella enterica

serotype Enteritidis to demonstrate the effectiveness of current sampling strategies. This

includes areas of the lettuce head most likely to be cross-contaminated during post-harvest

handling of minimally processed living lettuce. Further research is necessary to explore Glo

Germ™ products as a food safety training resource by promoting awareness of proper handling

of leafy greens. These studies contribute to identifying post-harvest risks to ensure safer living

lettuce. This research study suggests that water, and handling may be an important routes of

contamination for lettuce grown hydroponically in controlled greenhouse settings: amplification

of contamination and additional growth on the heads can occur during post-harvest handling,

especially under temperature abusive conditions. One research question to explore further is to

investigate the if the contamination risk is reduced when a large portion of the roots are removed

at harvest, and does this negatively affect the shelf life. This practice may significantly reduce

transfer of pathogens to edible leaves reducing contamination risks, yet maintain a good shelf life

stability and premium quality. Additionally the addition of an absorbent pad to the bottom of the

clamshell could reduce condensation and potentially transfer of droplets to the lettuce leaves.

90

Greenhouse production of vegetables is increasing in popularity, as the consumer demand

for fresh, locally sourced products in increasing. It is important to expand our understanding of

hydroponic production practices by small-scale producers, including post-harvest handling risks

that may be applicable to field and hydroponic produced leafy greens, so interventions can be

implemented at critical stages to reduce harmful pathogens. This establishes the need to

implement guidelines of safer post-harvest handling practices for commercial scale hydroponic

production of leafy greens. Food safety of leafy greens produced in a greenhouse hydroponic

setting can be greatly improved by setting guidelines that target practices that minimize risks

during post-harvest handling. In addition to practices that minimize risks, screening practices to

detect contaminated leafy greens are critical. To the best of our knowledge, there is no set

guideline or practice required by law regarding post-harvest handling of leafy greens in a

hydroponic production. Obtaining Good Agricultural Practices (GAPs) certification is strongly

encouraged to focus on the improvement of the microbiological quality of leafy greens. GAPs

are critical and fundamental preventative methods to ensure microbiological safety of leafy

greens by reducing human pathogen contaminations. These efforts on established practices and

risk management will ensure consumers a safer leafy greens supply, which improves the public

well being.